Combination therapies

ABSTRACT

The presently disclosed subject matter provides combination therapies for treating diseases or disorders, e.g., cancers. In particular, the present disclosure provides methods of treatment comprising administering genetically engineered cells and radiation.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of International Patent ApplicationNo. PCT/US2020/030690, filed Apr. 30, 2020, which claims priority toU.S. Provisional Patent Application Ser. No. 62/840,906, filed Apr. 30,2019, the contents of each of which are incorporated by reference in itsentirety, and to each of which priority is claimed.

2. SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on Oct. 28, 2021. Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing text file, identified as089333_0427_SL.txt, is 8,511 bytes and was created on Oct. 27, 2021. TheSequence Listing electronically filed herewith, does not extend beyondthe scope of the specification and thus does not contain new matter.

3. TECHNICAL FIELD

The present disclosure relates to combination therapies for treatingdiseases or disorders, e.g., cancers. In particular, the presentdisclosure provides methods of treatment comprising administeringgenetically engineered cells and radiation.

4. BACKGROUND

Radiation therapy (XRT) works locally though DNA damage of tumor cellsand can induce a systemic abscopal effect; that is, it can provide animmune mediated anti-tumor response outside the radiation field. Thisresponse is facilitated by release of neo-antigens that generate andshape a systemic TCR-mediated anti-tumor immune response (Sridharan etal., Br J Cancer (2016); 115:252-60; Tang et al., Cancer Immunol Res(2014); 2:831-8). Checkpoint blockade synergizes with concurrent XRT,likely because of this increased T-cell exposure to neo-antigens et al.,Oncoimmunology (2014); 3:e28780; Postow et al., N Engl J Med. (2012);366:925-31; Park et al., Cancer Immunol Res (2015); 3:610-9; Victor etal., Nature (2015); 520:373-7).

Various immunotherapy and/or cell therapy methods are available fortreating diseases and conditions. For example, adoptive cell therapies(including those involving the administration of cells expressingchimeric receptors specific for a disease or disorder of interest, suchas chimeric antigen receptors (CARs) and/or other recombinant antigenreceptors, as well as other adoptive immune cell and adoptive T celltherapies) can be effective in the treatment of cancer and otherdiseases and disorders. Alternative methods are needed, for example, toprovide increased efficacy and/or reduced cytokine release syndrome incertain patient populations and/or for treating certain diseases orconditions. Among the provided embodiments are methods and uses thataddress such needs.

5. SUMMARY OF THE INVENTION

Provided are methods and compositions for treatment of subjects havingor suspected of having a disease or condition, such as cancer or otherproliferative disorder. In certain embodiments, the method includes (a)administering to a subject having a disease or condition a dose of cellsexpressing a recombinant receptor that binds to an antigen; and (b)administering to said subject radiation, wherein initiation ofadministration of the radiation is no later than about two weeks afteradministration of the recombinant receptor-expressing cells.

In certain embodiments, initiation of administration of the radiation isno later than about one week after administration of the recombinantreceptor-expressing cells. In certain embodiments, initiation ofadministration of the radiation is between about 5 days and about 10days after administration of the recombinant receptor-expressing cells.In certain embodiments, the subject has not relapsed at the time of orimmediately prior to initiation of administration of the radiation.

In certain embodiments, the disease or condition is a tumor or a cancer.The disease or condition can be selected from the group consisting ofblood cancers, B cell malignancies, colon cancer, lung cancer, livercancer, breast cancer, prostate cancer, ovarian cancer, skin cancer,melanoma, bone cancer, brain cancer, ovarian cancer, epithelial cancers,renal cell carcinoma, pancreatic adenocarcinoma, cervical carcinoma,colorectal cancer, glioblastoma, neuroblastoma, Ewing sarcoma,medulloblastoma, osteosarcoma, synovial sarcoma, mesothelioma, andcombinations thereof. In certain embodiments, the blood cancer isselected from the group consisting of leukemia, lymphoma, chroniclymphocytic leukemia (CLL), acute-lymphoblastic leukemia (ALL), HodgkinLymphoma, non-Hodgkin's lymphoma, Waldenstrom's Macroglobulinemia, acutemyeloid leukemia, multiple myeloma, mantle cell lymphoma, and indolent Bcell lymphoma. In certain embodiments, the disease or condition ismultiple myeloma.

The antigen can be a tumor antigen or a pathogen antigen. In certainembodiments, the antigen is a tumor antigen. The tumor antigen can beselected from the group consisting of BCMA, GPRCSD, FcRL5, orphantyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22,mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor,CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, OEPHa2, Erb-B2,Erb-B3, Erb-B4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA,IL-22R-alpha, IL-13R-alpha2, KDR, kappa light chain, Lewis Y, L1-celladhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2DLigands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72,VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen,PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2,CD123, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin,and biotinylated molecules. In certain embodiments, the tumor antigen isBCMA.

In certain embodiments, the recombinant receptor is a T cell receptor(TCR) or a functional non-T cell receptor. In certain embodiments, therecombinant receptor is a chimeric antigen receptor (CAR).

The CAR can include an extracellular antigen-binding domain thatspecifically binds to the antigen and an intracellular signaling domain.In certain embodiments, the intracellular signaling domain comprises anintracellular domain of a CD3-zeta (CD3ζ) chain. The intracellularsignaling domain can further include a costimulatory signaling region.The costimulatory signaling region can include a signaling domain ofCD28 or a portion thereof, a signaling domain of 4-1BB or a portionthereof, a signaling domain of OX40 or a portion thereof, a signalingdomain of ICOS or a portion thereof, a signaling domain of DAP-10 or aportion thereof, or a combination thereof. In certain embodiments, thecostimulatory signaling region comprises a signaling domain of 4-1BB ora portion thereof.

The CAR can further include a transmembrane domain. The transmembranedomain can include a transmembrane domain of CD8 or a portion thereof,or a transmembrane domain of CD28 or a portion thereof. In certainembodiments, the transmembrane domain comprises a transmembrane domainof a CD8 or a portion thereof.

In certain embodiments, the extracellular antigen-binding domaincomprises a scFv. In certain embodiments, the extracellularantigen-binding domain comprises: a heavy chain variable region(“V_(H)”) CDR1 comprising the amino acid sequence set forth in SEQ IDNO: 1; a V_(H) CDR2 comprising the amino acid sequence set forth in SEQID NO: 2; a V_(H) CDR3 comprising the amino acid sequence set forth inSEQ ID NO: 3; a light chain variable region (“V_(L)”) CDR1 comprisingthe amino acid sequence set forth in SEQ ID NO: 4; a V_(L) CDR2comprising the amino acid sequence set forth in SEQ ID NO: 5; and aV_(L) CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.

In certain embodiments, the cell is a T cell. The T cell can be selectedfrom the group consisting of a cytotoxic T lymphocyte (CTL), aregulatory T cell, a tumor-infiltrating lymphocyte (TIL), and a NaturalKiller T (NKT) cell. The cell can be autologous or allogenic to thesubject.

In certain embodiments, the dose of cells comprises cells in an amountsufficient for reduction in burden of a disease or condition in thesubject. In certain embodiments, the method includes administering tothe subject a consecutive dose of the recombinant receptor-expressingcells after administration of a first dose of the recombinantreceptor-expressing cells.

The radiation can be selected from the group consisting of external beamradiation, a radiopharmaceutical agent, and a combination thereof. Incertain embodiments, the radiation is external beam radiation. Incertain embodiments, a total of at least about 10 Gy of radiation isadministered to a lesion site of the subject. In certain embodiments, atotal of between about 10 Gy and about 30 Gy of radiation isadministered to a lesion site of the subject. In certain embodiments, atotal of about 20 Gy of radiation is administered to a lesion site ofthe subject.

In certain embodiments, administration of the radiation andadministration of the recombinant receptor-expressing cells provide asynergistic abscopal effect. In certain embodiments, administration ofthe radiation and administration of the recombinant receptor-expressingcells provide delayed or reduced CRS-like response. In certainembodiments, administration of the radiation and administration of therecombinant receptor-expressing cells provide systemic expansion of newT-cell receptor (TCR) clone.

The present discourse also provides radiation and cells expressing arecombinant receptor that binds to an antigen are for use in a therapy,wherein initiation of administration of the radiation is no later thanabout two weeks after administration of the recombinantreceptor-expressing cells.

6. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F depict BCMA-targeted CAR T-cell therapy followed byradiation therapy (XRT) led to clinical response, expansion of TCRclonality, and CRS-like findings after XRT. The subject receivedconditioning therapy with cyclophosphamide and fludarabine followed byCAR T cells on day 0. Radiation therapy (XRT) took place over 10fractions between day 6 and day 20 (box). FIG. 1A shows pre-treatmentPET/CT scan showing extensive intra-osseous and extra-osseous FDG-aviddisease including soft tissue and pleural-based masses. FIG. 1B showsdecrease in M-spike commencing during XRT. FIG. 1C shows PET/CT 8 weekspost-therapy demonstrating resolution of MM lesions. FIG. 1D showsproduction of IL6 and CRP (pro-inflammatory markers associated withactive CAR T-cell function) peaked after the conclusion of XRT. FIG. 1Eshows daily maximum temperature curve revealed a fever at the time ofpeak IL6 and CRP. FIG. 1F shows TCR clonality analysis demonstratingexpansion of novel TCR clones. The subset of TCRs comprising newlyexpanding clones are shown over time.

FIGS. 2A and 2B depict local early response to radiation therapy. FIG.2A shows MRIs pre- (lef) and 4 weeks post- (right) CAR T cell therapy (1week post-conclusion of radiation therapy). FIG. 2B shows radiationfields. Conventionally fractioned radiation therapy was delivered to thethoracic spine (T1 to T8) over 5 days, followed by to the whole brain toC2 over 5 days. The total dose was 2000 cGy in 5 fractions to each site.

FIGS. 3A-3C depict additional inflammatory markers elevated in responseto CAR T cell therapy plus radiation therapy. FIG. 3A shows Ferritin.FIG. 3B shows D-dimer. FIG. 3C shows IL-10.

FIG. 4 depicts CAR T cells expanded and maintained persistence after theinitiation of high dose steroid taper and including through the periodof radiation therapy. CAR transgene as detected by PCR from peripheralblood.

FIG. 5 depicts persistence of CAR T cells around the time of expansionof TCR clonality and CRS-like findings after XRT. CAR T cells in thepatient's blood were assessed at 5 weeks after CAR T-cell therapy byflow cytometry with a fluorophore-labeled antibody recognizing thesurrogate transduction marker that is additionally expressed by the CARvector. Flow cytometry of peripheral blood mononuclear cells was gatedon viable CD3⁺ cells. The red box outlines cells positive for thetransduction marker (9.3% of circulating T cells). Note that thesepersistent gene-modified T cells were predominantly CD8⁺.

FIG. 6 depicts TCR repertoire of bone marrow mirrored that of peripheralblood. The TCR repertoire of bone marrow and peripheral bloodmononuclear cells from the same time point was evaluated. TCR diversitywas compared by Morisita's Overlap Index (C_(D)).

FIG. 7 depicts addition of radiation therapy to CAR T-cell therapyincreased TCR repertoire diversity over time. Heatmap of Morisita'sOverlap Indices (C_(D)) displays the degree of similarity between TCRrepertoire from peripheral blood samples over time. The index rangesfrom 0 (no overlap of TCR clonotypes) to 1 (all TCR clonotypes occur inthe same proportions in both samples).

FIG. 8 depicts highly expressed baseline TCR clones did not expand afterCAR T cell administration and radiation therapy. The frequency of thesubset of TCRs comprising TCR clones with baseline >2% frequency isshown over time.

7. DETAILED DESCRIPTION OF THE INVENTION

The presently disclosed subject matter provides methods of treatmentsinvolving the administration of genetically engineered cells andradiation to subjects having a disease or condition. The cells areengineered to express one or more recombinant receptor, for example achimeric antigen receptor (CAR).

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

For purposes of clarity of disclosure and not by way of limitation, thedetailed description is divided into the following subsections:

7.1. Definitions;

7.2. Methods for Treatments by Using Genetically Engineered Cells andRadiation;

7.3. Recombinant Receptors Expressed by the Cells;

7.4. Genetically Engineered Cells and Methods of Producing Cells;

7.5. Compositions and Formulations of Genetically Engineered Cells;

7.6. Dosing of the Genetically Engineered Cells;

7.7. Radiation Therapy;

7.8. Administration of Radiation Therapy;

7.9. Reduction in Disease Burden, Efficacy and Survival; and

7.10. Kits

7.1. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art.

As used herein, the term “about” or “approximately” means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” can mean within 3 or more than 3 standarddeviations, per the practice in the art. Alternatively, “about” can meana range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a givenvalue. Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, e.g., within5-fold or within 2-fold, of a value.

As used herein, the term “antibody” means not only intact antibodymolecules, but also fragments of antibody molecules that retainimmunogen-binding ability. Such fragments are also well known in the artand are regularly employed both in vitro and in vivo. Accordingly, asused herein, the term “antibody” means not only intact immunoglobulinmolecules but also the well-known active fragments F(ab′)₂, and Fab.F(ab′)₂, and Fab fragments that lack the Fe fragment of intact antibody,clear more rapidly from the circulation, and may have less non-specifictissue binding of an intact antibody (Wahl et al., J. Nucl. Med.24:316-325 (1983). As used herein, antibodies include whole nativeantibodies, bispecific antibodies; chimeric antibodies; Fab, Fab′,single chain V region fragments (scFv), fusion polypeptides, andunconventional antibodies. In certain embodiments, an antibody is aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain is comprisedof a heavy chain variable region (abbreviated herein as V_(H)) and aheavy chain constant (CH) region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as V_(L))and a light chain constant CL region. The light chain constant region iscomprised of one domain, CL. The V_(H) and V_(L) regions can be furthersub-divided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (C1 q) of the classicalcomplement system.

As used herein, “CDRs” are defined as the complementarity determiningregion amino acid sequences of an antibody which are the hypervariableregions of immunoglobulin heavy and light chains. See, e.g., Kabat etal., Sequences of Proteins of Immunological Interest, 4th U. S.Department of Health and Human Services, National Institutes of Health(1987). Generally, antibodies comprise three heavy chain and three lightchain CDRs or CDR regions in the variable region. CDRs provide themajority of contact residues for the binding of the antibody to theantigen or epitope. In certain embodiments, the CDRs regions aredelineated using the Kabat system (Kabat, E. A., et al. (1991) Sequencesof Proteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242). In certainembodiments, the CDRs are delineated using the Chothia numbering system(Chothia et al., J Mol Biol. (1987) 196:901-17). In certain embodiments,the CDRs are delineated using the AbM numbering system (Abhinandan etal., Mol. Immunol. 2008, 45, 3832-3839). In certain embodiments, theCDRs regions are delineated using the IMGT numbering system (accessibleathttp://www.imgt.org/IIVIGTScientificChart/Numbering/IMGTIGVLsuperfamily.html,http://www.imgt.org/IMGTindex/numbering.php).

As used herein, the term “single-chain variable fragment” or “scFv” is afusion protein of the variable regions of the heavy (V_(H)) and lightchains (V_(L)) of an immunoglobulin covalently linked to form aV_(H)::V_(L) heterodimer. The V_(H) and V_(L) are either joined directlyor joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 aminoacids), which connects the N-terminus of the V_(H) with the Cterminus ofthe V_(L), or the C-terminus of the V_(H) with the N-terminus of theV_(L). The linker is usually rich in glycine for flexibility, as well asserine or threonine for solubility.

As used herein, the term “linker” means a functional group (e.g.,chemical or polypeptide) that covalently attaches two or morepolypeptides or nucleic acids so that they are connected to one another.As used herein, a “peptide linker” refers to one or more amino acidsused to couple two proteins together (e.g., to couple V_(H) and V_(L)domains). Despite removal of the constant regions and the introductionof a linker, scFv proteins retain the specificity of the originalimmunoglobulin. Single chain Fv polypeptide antibodies can be expressedfrom a nucleic acid including V_(H)- and V_(L) encoding sequences asdescribed by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883,1988). See, also, U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,956,778; andU.S. Patent Publication Nos. 20050196754 and 20050196754. AntagonisticscFvs having inhibitory activity have been described (see, e.g., Zhao etal., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J CachexiaSarcopenia Muscle 2012 Aug. 12; Shieh et al., J Imunol 2009183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63;Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al.,Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 19952(10:31-40). Agonistic scFvs having stimulatory activity have beendescribed (see, e.g., Peter et al., J Bioi Chern 2003 25278(38):36740-7;Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit RevImmunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 20031638(3):257-66).

As used herein, “F(ab)” refers to a fragment of an antibody structurethat binds to an antigen but is monovalent and does not have a Fcportion, for example, an antibody digested by the enzyme papain yieldstwo F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constantregion; Fc region that does not bind to an antigen).

As used herein, “F(ab′)₂” refers to an antibody fragment generated bypepsin digestion of whole IgG antibodies, wherein this fragment has twoantigen binding (ab′) (bivalent) regions, wherein each (ab′) regioncomprises two separate amino acid chains, a part of a H chain and alight (L) chain linked by an S—S bond for binding an antigen and wherethe remaining H chain portions are linked together. A “F(ab′)2” fragmentcan be split into two individual Fab′ fragments.

As used herein, the term “vector” refers to any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.,which is capable of replication when associated with the proper controlelements and which can transfer gene sequences into cells. Thus, theterm includes cloning and expression vehicles, as well as viral vectorsand plasmid vectors. In certain embodiments, a vector refers to anucleic acid molecule capable of propagating another nucleic acid towhich it is linked. The term includes the vector as a self-replicatingnucleic acid structure as well as the vector incorporated into thegenome of a host cell into which it has been introduced. Certain vectorsare capable of directing the expression of nucleic acids to which theyare operatively linked. Such vectors are referred to herein as“expression vectors.”

As used herein, the term “expression vector” refers to a recombinantnucleic acid sequence, i.e. recombinant DNA molecule, containing adesired coding sequence and appropriate nucleic acid sequences necessaryfor the expression of the operably linked coding sequence in aparticular host organism. Nucleic acid sequences necessary forexpression in prokaryotes usually include a promoter, an operator(optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “affinity” is meant a measure of bindingstrength. Affinity can depend on the closeness of stereochemical fitbetween antibody combining sites and antigen determinants, on the sizeof the area of contact between them, and/or on the distribution ofcharged and hydrophobic groups. As used herein, the term “affinity” alsoincludes “avidity”, which refers to the strength of the antigen-antibodybond after formation of reversible complexes. Methods for calculatingthe affinity of an antibody for an antigen are known in the art,including, but not limited to, various antigen-binding experiments,e.g., functional assays (e.g., flow cytometry assay).

By “substantially identical” or “substantially homologous” is meant apolypeptide or a nucleic acid molecule exhibiting at least about 50%homologous or identical to a reference amino acid sequence (for example,any one of the amino acid sequences described herein) or a nucleotideacid sequence (for example, any one of the nucleic acid sequencesdescribed herein). In certain embodiments, such a sequence is at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 99%, or at least about 100% homologous oridentical to the amino acid sequence or the nucleotide sequence used forcomparison.

Sequence identity can be measured by using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e-3 and e-100 indicating a closely related sequence.

By “disease” is meant any condition, disease or disorder that damages orinterferes with the normal function of a cell, tissue, or organ, e.g.,neoplasia, and pathogen infection of cell.

By “modulate” is meant positively or negatively alter. Exemplarymodulations include a about 1%, about 2%, about 5%, about 10%, about25%, about 50%, about 75%, or about 100% change.

By “increase” is meant to alter positively by at least about 5%. Analteration may be by about 5%, about 10%, about 25%, about 30%, about50%, about 75%, about 100% or more.

By “reduce” is meant to alter negatively by at least about 5%. Analteration may be by about 5%, about 10%, about 25%, about 30%, about50%, about 75%, or even by about 100%.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide is purified if it is substantially free of cellularmaterial, viral material, or culture medium when produced by recombinantDNA techniques, or chemical precursors or other chemicals whenchemically synthesized. Purity and homogeneity are typically determinedusing analytical chemistry techniques, for example, polyacrylamide gelelectrophoresis or high performance liquid chromatography. The term“purified” can denote that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. For a protein that canbe subjected to modifications, for example, phosphorylation orglycosylation, different modifications may give rise to differentisolated proteins, which can be separately purified.

By “isolated cell” is meant a cell that is separated from the molecularand/or cellular components that naturally accompany the cell.

The term “antigen-binding domain” as used herein refers to a domaincapable of specifically binding a particular antigenic determinant or aset of antigenic determinants present on a cell.

By “neoplasm” is meant a disease characterized by the pathologicalproliferation of a cell or tissue and its subsequent migration to orinvasion of other tissues or organs. The growth of neoplasm is typicallyuncontrolled and progressive, and occurs under conditions that would notelicit, or would cause cessation of, multiplication of normal cells.Neoplasm can affect a variety of cell types, tissues, or organs,including but not limited to an organ selected from the group consistingof bladder, bone, brain, breast, cartilage, glia, esophagus, fallopiantube, gallbladder, heart, intestines, kidney, liver, lung, lymph node,nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin,spinal cord, spleen, stomach, testes, thymus, thyroid, trachea,urogenital tract, ureter, urethra, uterus, and vagina, or a tissue orcell type thereof. Neoplasia include cancers, such as sarcomas,carcinomas, or plasmacytomas (malignant tumor of the plasma cells).

By “recognize” is meant selectively binds to a target. A T cell thatrecognizes a tumor can expresses a CAR that binds to a tumor antigen.

By “specifically binds” is meant a polypeptide or a fragment thereofthat recognizes and binds to a biological molecule of interest (e.g., apolypeptide), but which does not substantially recognize and bind othermolecules in a sample, for example, a biological sample, which naturallyincludes a presently disclosed polypeptide.

The term “tumor antigen” as used herein includes a tumor-specificantigen (TSA), which is present only on tumor cells and not on any othercell, and tumor-associated antigen (TAA), which is present on some tumorcells and also some normal cells. In certain embodiments, the tumorantigen is a TSA. In certain embodiments, the tumor antigen is uniquelyor differentially expressed on a tumor cell compared to a normal ornon-neoplastic cell. In certain embodiments, a tumor antigen includesany polypeptide expressed by a tumor that is capable of activating orinducing an immune response via an antigen recognizing receptor (e.g.,mesothelin) or capable of suppressing an immune response viareceptor-ligand binding (e.g., CD47, PD-L1/L2, B7.1/2).

The terms “comprises”, “comprising”, and are intended to have the broadmeaning ascribed to them in U.S. Patent Law and can mean “includes”,“including” and the like.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to complete or partial amelioration orreduction of a disease or condition or disorder, or a symptom, adverseeffect or outcome, or phenotype associated therewith. Desirable effectsof treatment include, but are not limited to, preventing occurrence orrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease, preventingmetastasis, decreasing the rate of disease progression, amelioration orpalliation of the disease state, and remission or improved prognosis.The terms do not imply complete curing of a disease or completeelimination of any symptom or effect(s) on all symptoms or outcomes.

As used herein, “delaying development of a disease” means to defer,hinder, slow, retard, stabilize, suppress and/or postpone development ofthe disease (such as cancer). This delay can be of varying lengths oftime, depending on the history of the disease and/or individual beingtreated. As is evident to one skilled in the art, a sufficient orsignificant delay can, in effect, encompass prevention, in that theindividual does not develop the disease. For example, a late stagecancer, such as development of metastasis, may be delayed.

“Preventing,” as used herein, includes providing prophylaxis withrespect to the occurrence or recurrence of a disease in a subject thatmay be predisposed to the disease but has not yet been diagnosed withthe disease. In certain embodiments, the provided cells and compositionsare used to delay development of a disease or to slow the progression ofa disease.

As used herein, to “suppress” a function or activity is to reduce thefunction or activity when compared to otherwise same conditions exceptfor a condition or parameter of interest, or alternatively, as comparedto another condition. For example, cells that suppress tumor growthreduce the rate of growth of the tumor compared to the rate of growth ofthe tumor in the absence of the cells.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,cells, or composition, in the context of administration, refers to anamount effective, at dosages/amounts and for periods of time necessary,to achieve a desired result, such as a therapeutic or prophylacticresult.

A “therapeutically effective amount” of an agent, e.g., a pharmaceuticalformulation or cells, refers to an amount effective, at dosages and forperiods of time necessary, to achieve a desired therapeutic result, suchas for treatment of a disease, condition, or disorder, and/orpharmacokinetic or pharmacodynamic effect of the treatment. Thetherapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the subject, and thepopulations of cells administered. In certain embodiments, the providedmethods involve administering the cells and/or compositions at effectiveamounts, e.g., therapeutically effective amounts.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount. In the context of lower tumor burden, theprophylactically effective amount in some aspects will be higher thanthe therapeutically effective amount.

An “individual” or “subject” herein is a vertebrate, such as a human ornon-human animal, for example, a mammal. Mammals include, but are notlimited to, humans, primates, farm animals, sport animals, rodents andpets. Non-limiting examples of non-human animal subjects include rodentssuch as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats;sheep; pigs; goats; cattle; horses; and non-human primates such as apesand monkeys. In certain embodiments, the primate is a monkey or an ape.The subject can be male or female and can be any suitable age, includinginfant, juvenile, adolescent, adult, and geriatric subjects. In certainembodiments, the subject is a non-primate mammal, such as a rodent.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,“a” or “an” means “at least one” or “one or more.”

As used herein, a “composition” refers to any mixture of two or moreproducts, substances, or compounds, including cells. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

Throughout this disclosure, various aspects of the claimed subjectmatter are presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theclaimed subject matter. Accordingly, the description of a range shouldbe considered to have specifically disclosed all the possible sub-rangesas well as individual numerical values within that range. For example,where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the claimed subject matter. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the claimed subjectmatter, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe claimed subject matter. This applies regardless of the breadth ofthe range.

7.2. METHODS FOR TREATMENTS BY USING GENETICALLY ENGINEERED CELLS ANDRADIATION

The presently disclosed subject matter is based on the discovery thatgenetically engineered cells (e.g., chimeric antigen receptor(CAR)-expressing T cells) can be used in combination with a radiationsource, such as external beam irradiation or a radioisotope, such as aradiopharmaceutical, to provide therapeutically effective anticancereffects.

Furthermore, an unexpected synergistic effect between the geneticallyengineered cells and the radiation source results in an enhanced orsynergistic therapeutic effect, wherein the combined effect is greaterthan the additive effect resulting from administration of the cells andradiation each at a therapeutic dose. These observations support thatgenetically engineered cells (e.g., chimeric CAR-expressing T cells) canbe used in combination with radiotherapy for treating diseases ordisorders (e.g., cancers).

The present disclosure is at least based on the observation that thecombination of a first treatment procedure that includes administrationof an effective amount of genetically engineered cells (e.g.,CAR-expressing T cells), as described herein, and a second treatmentprocedure using radiation treatment, as described herein, to a subjectin need thereof can provide a) a synergistic abscopal-like response,therapeutically effective anti-cancer effects, delayed or reducedCRS-like response, and/or systemic expansion of new T-cell receptor(TCR) clone (e.g., in peripheral blood).

Provided are methods for treating diseases or conditions, includingvarious cancers and tumors. In certain embodiments, the method includesadministering (a) radiation and (b) cells expressing recombinantreceptors designed to recognize and/or specifically bind to a molecule(e.g., an antigen) associated with the disease or condition to betreated and result in a response, such as an immune response againstsuch molecule upon binding to such molecule. The recombinant receptorsinclude chimeric receptors, e.g., chimeric antigen receptors (CARs), andother transgenic antigen receptors including transgenic T cell receptors(TCRs).

In certain embodiments, the methods are advantageous in their ability totreat subjects having certain diseases or conditions such as cancers(e.g., multiple myeloma) by initiating the radiation therapy soon afterthe subject has received the cells expressing the recombinant receptor(referred to as “the recombinant receptor-expressing cells”). Forexample, in certain embodiments, the method comprises initiating theradiation therapy no later than about two weeks after administration ofthe recombinant receptor-expressing cells. In certain embodiments, themethod comprises initiating the radiation therapy no later than aboutone week after administration of the recombinant receptor-expressingcells. In certain embodiments, the method comprises initiating theradiation therapy between about 5 days and about 10 days afteradministration of the recombinant receptor-expressing cells. In certainembodiments, the method comprises initiating the radiation therapy about5 days after administration of the recombinant receptor-expressingcells. In certain embodiments, the method comprises initiating theradiation therapy 6 days after administration of the recombinantreceptor-expressing cells.

In certain embodiments, the subject has not relapsed at the time of orimmediately prior to initiation of the radiation therapy.

In certain embodiments, the method includes administration of therecombinant receptor-expressing cells or a composition comprising thecells to a subject, tissue, or cell, such as one having, at risk for, orsuspected of having the disease, condition or disorder. In certainembodiments, the recombinant receptor-expressing cells or compositionscomprising thereof are administered to a subject having the particulardisease or condition to be treated, e.g., via adoptive cell therapy,such as adoptive T cell therapy. In certain embodiments, the recombinantreceptor-expressing cells or compositions comprising thereof areadministered to the subject, such as a subject having or at risk for thedisease or condition, ameliorate one or more symptom of the disease orcondition.

The disease or condition that is treated is generally one with whichexpression of the target antigen is associated and/or involved in theetiology thereof, e.g. in which the antigen causes, exacerbates orotherwise is involved in such disease, condition, or disorder or issimply a marker of or is overexpressed or uniquely expressed on cells ofthe disease or disorder. Exemplary diseases and conditions can include,but are not limited to, diseases or conditions associated withmalignancy or transformation of cells (e.g. cancer). Exemplary antigens,which include antigens associated with various diseases and conditionsthat can be treated, are described above. In certain embodiments, theCAR or TCR specifically binds to an antigen associated with the diseaseor condition.

Among the diseases, conditions, and disorders are tumors, includingsolid tumors, hematologic malignancies (e.g., blood cancers), andmelanomas, and including localized and metastatic tumors. In certainembodiments, the subject has an infectious disease, such as infectionwith a virus or other pathogen, e.g., HIV, HCV, HBV, CMV, and parasiticdisease.

In certain embodiments, the disease or condition is a tumor, cancer,malignancy, neoplasm, or other proliferative disease or disorder. Suchdiseases include but are not limited to, blood cancers (including, butnot limited to, leukemia, chronic lymphocytic leukemia (CLL),acute-lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (e.g.,refractory follicular lymphoma), Waldenstrom's Macroglobulinemia,Hodgkin's lymphoma, acute myeloid leukemia, multiple myeloma, mantlecell lymphoma, and indolent B cell lymphoma), B cell malignancies, coloncancer, lung cancer, liver cancer, breast cancer, prostate cancer,ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer,ovarian cancer, epithelial cancers, renal cell carcinoma, pancreaticadenocarcinoma, cervical carcinoma, colorectal cancer, glioblastoma,neuroblastoma, Ewing sarcoma, medulloblastoma, osteosarcoma, synovialsarcoma, and/or mesothelioma. In certain embodiments, the subject hasblood cancer. In certain embodiments, the disease is multiple myeloma.

In certain embodiments, the disease or condition is an infectiousdisease or condition, such as, but not limited to, viral, retroviral,bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus(CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus. In certainembodiments, the disease or condition is an autoimmune or inflammatorydisease or condition, such as arthritis, e.g., rheumatoid arthritis(RA), Type I diabetes, systemic lupus erythematosus (SLE), inflammatorybowel disease, psoriasis, scleroderma, autoimmune thyroid disease,Grave's disease, Crohn's disease, multiple sclerosis, asthma, and/or adisease or condition associated with transplant.

In certain embodiments, the antigen associated with the disease ordisorder is selected from the group consisting of BCMA, orphan tyrosinekinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin,CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24,CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, OEPHa2, Erb-B2, Erb-B3,Erb-B4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA,IL-22R-alpha, IL-13R-alpha2, KDR, kappa light chain, Lewis Y, L1-celladhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2DLigands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72,VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen,PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2,CD123, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin(e.g., cyclin A1 (CCNA1)), biotinylated molecules, molecules expressedby HIV, HCV, HBV, and other pathogens. In certain embodiments, theantigen is BCMA.

In certain embodiments, the provided methods can enhance overallsurvival of subjects in which subjects exhibit a particular level ofdisease burden at the time of treatment such as morphologic or minimaldisease.

7.3. RECOMBINANT RECEPTORS EXPRESSED BY THE CELLS

In certain embodiments, the cells for use in or administered inconnection with the provided methods contain or are engineered toinclude a recombinant receptor (or an engineered receptor), e.g., anengineered antigen receptor, such as a chimeric antigen receptor (CAR),or a T cell receptor (TCR). Also provided are populations of such cells,compositions containing such cells and/or enriched for such cells, suchas in which cells of a certain type such as T cells or CD8⁺ or CD4⁺cells are enriched or selected. Among the compositions arepharmaceutical compositions and formulations for administration, such asfor adoptive cell therapy. Also provided are therapeutic methods foradministering the cells and compositions to subjects, e.g., patients, inaccord with the provided methods.

In certain embodiments, the cells include one or more nucleic acidsintroduced via genetic engineering, and thereby express recombinant orgenetically engineered products of such nucleic acids. In certainembodiments, gene transfer is accomplished by first stimulating thecells, such as by combining it with a stimulus that induces a responsesuch as proliferation, survival, and/or activation, e.g., as measured byexpression of a cytokine or activation marker, followed by transductionof the activated cells, and expansion in culture to numbers sufficientfor clinical applications.

The cells generally express recombinant receptors, such as antigenreceptors including functional non-TCR antigen receptors, e.g., chimericantigen receptors (CARs), and other antigen-binding receptors such astransgenic T cell receptors (TCRs).

7.3.1. CHIMERIC ANTIGEN RECEPTORS (CARS

In certain embodiments of the provided methods and uses, the recombinantreceptor is a chimeric antigen receptor (CAR), which comprises anextracellular antigen-binding domain that provides specificity for anantigen (e.g., a tumor antigen) and an intracellular signaling domain.In certain embodiments, the CAR comprises a transmembrane domain. Incertain embodiments, the intracellular signaling domain is an activatingintracellular domain portion, such as a T cell activating domain,providing a primary activation signal. In certain embodiments, theintracellular signaling domain includes a costimulatory signaling regionto facilitate effector functions. In certain embodiments, therecombinant receptors when genetically engineered into immune cells canmodulate T cell activity, and, in some cases, can modulate T celldifferentiation or homeostasis, thereby resulting in geneticallyengineered cells with improved longevity, survival and/or persistence invivo, such as for use in adoptive cell therapy methods.

Exemplary antigen receptors, including CARs, and methods for engineeringand introducing such receptors into cells, include those described, forexample, in international patent application publication numbersWO200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321,WO2013/071154, WO2013/123061 U.S. patent application publication numbersUS2002131960, US2013287748, US20130149337, U.S. Pat. Nos. 6,451,995,7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319,7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118,and European patent application number EP2537416, and/or those describedby Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila etal. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol.,2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 Mar. 18(2): 160-75.In certain embodiments, the recombinant receptors include a CAR asdescribed in U.S. Pat. No. 7,446,190, and those described inInternational Patent Application Publication No.: WO/2014055668 A1.Examples of the CARs include CARs as disclosed in any of theaforementioned publications, such as WO2014031687, U.S. Pat. Nos.8,339,645, 7,446,179, US 2013/0149337, U.S. Pat. Nos. 7,446,190,8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology,10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701;and Brentjens et al., Sci Transl Med. 2013 5(177). See alsoWO2014031687, U.S. Pat. Nos. 8,339,645, 7,446,179, US 2013/0149337, U.S.Pat. Nos. 7,446,190, and 8,389,282.

The recombinant receptors, such as CARs, generally include anextracellular antigen-binding domain, such as a portion of an antibodymolecule, generally a variable heavy (V_(H)) chain region and/orvariable light (V_(L)) chain region of the antibody. In certainembodiments, the extracellular antigen-binding domain comprises a scFv.The scFv can be a human, murine or humanized scFv. In certainembodiments, the scFv is a human scFv. In certain embodiments, theextracellular antigen-binding domain comprises a Fab, which isoptionally crosslinked. In certain embodiments, the extracellularantigen-binding domain comprises a F(ab)₂. In certain embodiments, anyof the foregoing molecules may be comprised in a fusion protein with aheterologous sequence to form the extracellular antigen-binding domain.In certain embodiments, the scFv is identified by screening scFv phagelibrary with an antigen-Fc fusion protein. The scFv can be derived froma mouse bearing human V_(L) and/or V_(H) genes. The scFv can also besubstituted with a camelid Heavy chain (e.g., VHH, from camel, lama,etc.) or a partial natural ligand for a cell surface receptor.

In certain embodiments, the antigen targeted by the receptor is apolypeptide. In certain embodiments, it is a carbohydrate or othermolecule. In certain embodiments, the antigen is selectively expressedor overexpressed on cells of the disease or condition, e.g., the tumoror pathogenic cells, as compared to normal or non-targeted cells ortissues. In certain embodiments, the antigen is expressed on normalcells and/or is expressed on the engineered cells. In certainembodiments, the antigen is a tumor antigen or a pathogen antigen.

In certain embodiments, antigens targeted by the receptors include, butnot limited to, BCMA, G-protein coupled receptor (e.g., a G-proteincoupled receptor family C group 5 member D (GPRCSD)), Fc Receptor-like 5(FcRL5), orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM,CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen,anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2,EGP-4, OEPHa2, Erb-B2, Erb-B3, Erb-B4, FBP, fetal acethycholine ereceptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappalight chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin,MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gp100, oncofetalantigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostatespecific antigen, PSMA, Her2/neu, estrogen receptor, progesteronereceptor, ephrinB2, CD123, c-Met, GD-2, and MAGE A3, CE7, Wilms Tumor 1(WT-1), a cyclin (e.g., cyclin A1 (CCNA1)), and biotinylated molecules.In certain embodiments, antigens targeted by the receptors include, butnot limited to, molecules expressed by HIV, HCV, HBV, and otherpathogens.

In certain embodiments, the CAR binds to a pathogen-specific antigen. Incertain embodiments, the CAR is specific for viral antigens (such asHIV, HCV, HBV, etc.), bacterial antigens, and/or parasitic antigens.

In certain embodiments, the recombinant receptor (e.g., a CAR) targetsBCMA (e.g., human BCMA). In certain embodiments, the cells of thepresently disclosed subject matter express a BCMA-targeted CAR disclosedin the International Patent Application Publication No. WO2016/090320,which is incorporated by reference in its entirety. In certainembodiments, the cells of the presently disclosed subject matter expressthe BCMA-targeted CAR disclosed in Smith et al., Molecular Therapy(2018); 26(6):1447-1456), which is incorporated by reference in itsentirety. In certain embodiments, the extracellular antigen-bindingdomain of the BCMA-targeted CAR comprises an antigen-binding fragment(e.g., scFv) disclosed in WO2016/090320 and WO2016/090327, which areincorporated by reference in their entireties.

In certain embodiments, the CAR includes an extracellularantigen-binding domain that includes an antibody or a fragment thereof.In certain embodiments, the CAR includes an extracellularantigen-binding domain and an intracellular signaling domain. In certainembodiments, the extracellular antigen-binding domain includes a scFv.

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a heavy chain variable region(“V_(H)”) CDR1 comprising the amino acid sequence set forth in SEQ IDNO: 1; a V_(H) CDR2 comprising the amino acid sequence set forth in SEQID NO: 2; a V_(H) CDR3 comprising the amino acid sequence set forth inSEQ ID NO: 3; a light chain variable region (“V_(L)”) CDR1 comprisingthe amino acid sequence set forth in SEQ ID NO: 4; a V_(L) CDR2comprising the amino acid sequence set forth in SEQ ID NO: 5; and aV_(L) CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.In certain embodiments, the CDRs are identified according to the Kabatnumbering system. SEQ ID Nos: 1-6 are provided below.

[SEQ ID NO: 1] SYWIG [SEQ ID NO: 2] IIYPGDSDTRYSPSFQG [SEQ ID NO: 3]YSGSFDN [SEQ ID NO: 4] SGTSSNIGSHSVN [SEQ ID NO: 5] TNNQRPS[SEQ ID NO: 6] AAWDGSLNGLV

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a V_(H) CDR1 comprising the aminoacid sequence set forth in SEQ ID NO: 7; a V_(H) CDR2 comprising theamino acid sequence set forth in SEQ ID NO: 8; a V_(H) CDR3 comprisingthe amino acid sequence set forth in SEQ ID NO: 3; a V_(L) CDR1comprising the amino acid sequence set forth in SEQ ID NO: 4; a V_(L)CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and aV_(L) CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.In certain embodiments, the CDRs are identified according to the Chothianumbering system. SEQ ID Nos: 7 and 8 are provided below.

[SEQ ID NO: 7] GYSFTSY [SEQ ID NO: 8] YPGDSD

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a V_(H) CDR1 comprising the aminoacid sequence set forth in SEQ ID NO: 9; a V_(H) CDR2 comprising theamino acid sequence set forth in SEQ ID NO: 10; a V_(H) CDR3 comprisingthe amino acid sequence set forth in SEQ ID NO: 3; a V_(L) CDR1comprising the amino acid sequence set forth in SEQ ID NO: 4; a V_(L)CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and aV_(L) CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.In certain embodiments, the CDRs are identified according to the AbMnumbering system. SEQ ID Nos: 9 and 10 are provided below.

[SEQ ID NO: 9] GYSFTSYWIG [SEQ ID NO: 10] IIYPGDSDTR

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a V_(H) comprising an amino acidsequence that is at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, atleast about 86%, at least about 87%, at least about 88%, at least about89%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99% or atleast about 100% homologous or identical to the amino acid sequence setforth in SEQ ID NO: 11. In certain embodiments, the CAR comprises anextracellular antigen-binding domain that comprises: a V_(H) comprisingthe amino acid sequence set forth in SEQ ID NO: 11. SEQ ID NO: 11 isprovided below.

[SEQ ID NO: 11] EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGHVTISADKSISTAYLQWSSLKASDTAMYYCARYS GSFDNWGQGTLVTVSS

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a V_(L) comprising an amino acidsequence that is at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, atleast about 86%, at least about 87%, at least about 88%, at least about89%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99% or atleast about 100% homologous or identical to the amino acid sequence setforth in SEQ ID NO: 12. In certain embodiments, the CAR comprises anextracellular antigen-binding domain that comprises: a V_(H) comprisingthe amino acid sequence set forth in SEQ ID NO: 12. SEQ ID NO: 12 isprovided below.

[SEQ ID NO: 12] SYELTQPPSASGTPGQRVTMSCSGTSSNIGSHSVNWYQQLPGTAPKLLIYTNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDGSLNGLV FGGGTKLTVLG

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a V_(H) CDR1 comprising the aminoacid sequence set forth in SEQ ID NO: 13; a V_(H) CDR2 comprising theamino acid sequence set forth in SEQ ID NO: 14; a V_(H) CDR3 comprisingthe amino acid sequence set forth in SEQ ID NO: 15; a V_(L) CDR1comprising the amino acid sequence set forth in SEQ ID NO: 16; a V_(L)CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17; anda V_(L) CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, the CDRs are identified according to theKabat numbering system. SEQ ID Nos: 13-18 are provided below.

[SEQ ID NO: 13] DYYVY [SEQ ID NO: 14] WINPNSGGTNYAQKFQG [SEQ ID NO: 15]SQRDGYMDY [SEQ ID NO: 16] TGTSSDVG [SEQ ID NO: 17] EDSKRPS[SEQ ID NO: 18] SSNTRSSTLV

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a V_(H) CDR1 comprising the aminoacid sequence set forth in SEQ ID NO: 19; a V_(H) CDR2 comprising theamino acid sequence set forth in SEQ ID NO: 20; a V_(H) CDR3 comprisingthe amino acid sequence set forth in SEQ ID NO: 15; a V_(L) CDR1comprising the amino acid sequence set forth in SEQ ID NO: 16; a V_(L)CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17; anda V_(L) CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, the CDRs are identified according to theChothia numbering system. SEQ ID Nos: 19 and 20 are provided below.

[SEQ ID NO: 19] GYTFIDY [SEQ ID NO: 20] NPNSGG

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a V_(H) CDR1 comprising the aminoacid sequence set forth in SEQ ID NO: 21; a V_(H) CDR2 comprising theamino acid sequence set forth in SEQ ID NO: 22; a V_(H) CDR3 comprisingthe amino acid sequence set forth in SEQ ID NO: 15; a V_(L) CDR1comprising the amino acid sequence set forth in SEQ ID NO: 16; a V_(L)CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17; anda V_(L) CDR3 comprising the amino acid sequence set forth in SEQ ID NO:18. In certain embodiments, the CDRs are identified according to the AbMnumbering system. SEQ ID Nos: 21 and 22 are provided below.

[SEQ ID NO: 21] GYTFIDYYVY [SEQ ID NO: 22] WINPNSGGTN

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a V_(H) comprising an amino acidsequence that is at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, atleast about 86%, at least about 87%, at least about 88%, at least about89%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99% or atleast about 100% homologous or identical to the amino acid sequence setforth in SEQ ID NO: 23. In certain embodiments, the CAR comprises anextracellular antigen-binding domain that comprises: a V_(H) comprisingthe amino acid sequence set forth in SEQ ID NO: 23. SEQ ID NO: 23 isprovided below.

[SEQ ID NO: 23] EVQLVQSGAEMKKPGASLKLSCKASGYTFIDYYVYWMRQAPGQGLESMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAMYYCARSQ RDGYMDYWGQGTLVTVSS

In certain embodiments, the CAR comprises an extracellularantigen-binding domain that comprises: a V_(L) comprising an amino acidsequence that is at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, atleast about 86%, at least about 87%, at least about 88%, at least about89%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99% or atleast about 100% homologous or identical to the amino acid sequence setforth in SEQ ID NO: 24. In certain embodiments, the CAR comprises anextracellular antigen-binding domain that comprises: a V_(H) comprisingthe amino acid sequence set forth in SEQ ID NO: 24. SEQ ID NO: 24 isprovided below.

[SEQ ID NO: 24] QSALTQPASVSASPGQSIAISCTGTSSDVGWYQQHPGKAPKLMIYEDSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSNTRSSTLVFGGGTK LTVLG

In certain embodiments, the antibody portion of the recombinantreceptor, e.g., CAR, further includes at least a portion of animmunoglobulin constant region, such as a hinge region, e.g., an IgG4hinge region, and/or a CH1/CL and/or Fc region. In certain embodiments,the constant region or portion is of a human IgG, such as IgG4 or IgG1.In certain embodiments, the portion of the constant region serves as aspacer region between the antigen-recognition component, e.g., scFv, andtransmembrane domain. The spacer can be of a length that provides forincreased responsiveness of the cell following antigen binding, ascompared to in the absence of the spacer. Exemplary spacers include, butare not limited to, those described in Hudecek et al. (2013) Clin.Cancer Res., 19:3153, international patent application publicationnumber WO2014031687, U.S. Pat. No. 8,822,647 or published app. No.US2014/0271635.

In certain embodiments, the intracellular signaling domain is linkeddirectly or indirectly to the extracellular antigen-binding domain. Incertain embodiments, the CAR includes a transmembrane domain linking theextracellular antigen-binding domain and the intracellular signalingdomain. In certain embodiments, the intracellular signaling domaincomprises an ITAM. For example, in certain embodiments, theextracellular antigen-binding domain is linked to one or moreintracellular signaling components, such as signaling components thatmimic activation through an antigen receptor complex, such as a TCRcomplex, in the case of a CAR, and/or signal via another cell surfacereceptor.

In certain embodiments, a transmembrane domain that naturally isassociated with one of the domains in the receptor, e.g., CAR, is used.In certain embodiments, the transmembrane domain is selected or modifiedby amino acid substitution to avoid binding of such domains to thetransmembrane domains of the same or different surface membrane proteinsto minimize interactions with other members of the receptor complex.

In certain embodiments, the transmembrane domain is derived either froma natural or from a synthetic source. Where the source is natural, thedomain, in certain embodiments is derived from any membrane-bound ortransmembrane protein. Transmembrane regions include those derived from(i.e. comprise at least the transmembrane region(s) of) the alpha, betaor zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD154. Alternatively, in certain embodiments, the transmembrane domain issynthetic. In certain embodiments, the synthetic transmembrane domaincomprises predominantly hydrophobic residues such as leucine and valine.In certain embodiments, a triplet of phenylalanine, tryptophan andvaline is be found at each end of a synthetic transmembrane domain. Incertain embodiments, the linkage is by linkers, spacers, and/ortransmembrane domain(s). In certain embodiments, the transmembranedomain includes a transmembrane portion of CD28. In certain embodiments,the transmembrane domain includes a transmembrane portion of CD8.

In certain embodiments, the extracellular antigen-binding domain andtransmembrane domain can be linked directly or indirectly. In certainembodiments, the extracellular antigen-binding domain and transmembraneare linked by a spacer, such as any described herein. In certainembodiments, the recombinant receptor (e.g., CAR) includes extracellularportion of the molecule from which the transmembrane domain is derived,such as a CD28 extracellular portion, or a CD8 extracellular portion.

Among the intracellular signaling domains are those that mimic orapproximate a signal through a natural antigen receptor, a signalthrough such a receptor in combination with a costimulatory receptor,and/or a signal through a costimulatory receptor alone. In certainembodiments, a short oligo- or polypeptide linker, for example, a linkerof between 2 and 10 amino acids in length, such as one containingglycines and serines, e.g., glycine-serine doublet, is present and formsa linkage between the transmembrane domain and the cytoplasmic signalingdomain of the CAR.

In certain embodiments, T cell activation is described as being mediatedby two classes of cytoplasmic signaling sequences: those that initiateantigen-dependent primary activation through the TCR (primarycytoplasmic signaling sequences), and those that act in anantigen-independent manner to provide a secondary or co-stimulatorysignal (secondary cytoplasmic signaling sequences). In certainembodiments, the CAR includes one or both of such signaling components.

The recombinant receptor, e.g., the CAR, generally includes at least oneintracellular signaling component. In certain embodiments, the CARincludes a primary cytoplasmic signaling sequence that regulates primaryactivation of the TCR complex. Primary cytoplasmic signaling sequencesthat act in a stimulatory manner may contain signaling motifs which areknown as immunoreceptor tyrosine-based activation motifs or ITAMs.Examples of ITAM containing primary cytoplasmic signaling sequencesinclude those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3delta and CD3 epsilon. In certain embodiments, cytoplasmic signalingmolecule(s) in the CAR contain(s) a cytoplasmic signaling domain,portion thereof, or sequence derived from CD3 zeta.

In certain embodiments, the recombinant receptor includes anintracellular component of a TCR complex, such as a TCR CD3 chain thatmediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus,in certain embodiments, the antigen-binding portion is linked to one ormore cell signaling modules. In certain embodiments, the cell signalingmodules include a CD3 transmembrane domain, a CD3 intracellularsignaling domain, and/or other CD transmembrane domains. In certainembodiments, the recombinant receptor, e.g., CAR, further includes aportion of one or more additional molecules such as Fc receptor γ, CD8,CD4, CD25, or CD16. For example, in certain embodiments, the CAR orother chimeric receptor includes a chimeric molecule between CD3-zeta(CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.

In certain embodiments, upon ligation of the CAR or other recombinantreceptor, the cytoplasmic domain or intracellular signaling domain ofthe receptor activates at least one of the normal effector functions orresponses of the immune cell, e.g., T cell engineered to express theCAR. For example, in certain embodiments, the CAR induces a function ofa T cell such as cytolytic activity or T-helper activity, such assecretion of cytokines or other factors. In certain embodiments, atruncated portion of an intracellular signaling domain of an antigenreceptor component or costimulatory molecule is used in place of anintact immunostimulatory chain, for example, if it transduces theeffector function signal. In certain embodiments, the intracellularsignaling domain or domains include the cytoplasmic sequences of the Tcell receptor (TCR), and in certain embodiments also those ofco-receptors that in the natural context act in concert with suchreceptors to initiate signal transduction following antigen receptorengagement.

In the context of a natural TCR, full activation generally requires notonly signaling through the TCR, but also a costimulatory signal. Thus,in certain embodiments, to promote full activation, a component forgenerating secondary or co-stimulatory signal is also included in theCAR. In certain embodiments, the CAR does not include a component forgenerating a costimulatory signal. In certain embodiments, an additionalCAR is expressed in the same cell and provides the component forgenerating the secondary or costimulatory signal.

In certain embodiments, the CAR includes both the activating andcostimulatory components. In certain embodiments, the intracellularsignaling domain of the CAR comprises at least one co-stimulatorysignaling region. In certain embodiments, the at least oneco-stimulatory signaling region comprises an intracellular domain of acostimulatory molecule or a portion thereof. Non-limiting examples ofcostimulatory molecules include CD28, 4-1BB, OX40, DAP10, and ICOS. Incertain embodiments, the costimulatory molecule is CD28. In certainembodiments, the costimulatory molecule is 4-1BB.

In certain embodiments, the activating domain is included within oneCAR, whereas the costimulatory component is provided by another CARrecognizing another antigen. In certain embodiments, the CARs includeactivating or stimulatory CARs, costimulatory CARs, both expressed onthe same cell (see WO2014/055668). In certain embodiments, the cellsinclude one or more stimulatory or activating CAR and/or a costimulatoryCAR. In certain embodiments, the cells further include inhibitory CARs(“iCARs”, see Fedorov et al., Sci. Transl. Medicine (2013); 5:215) as aCAR recognizing an antigen other than the one associated with and/orspecific for the disease or condition whereby an activating signaldelivered through the disease-targeting CAR is diminished or inhibitedby binding of the inhibitory CAR to its ligand, e.g., to reduceoff-target effects.

In certain embodiments, the intracellular signaling domain comprises aCD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta)intracellular domain. In certain embodiments, the intracellularsignaling domain comprises a 4-1BB transmembrane and signaling domainlinked to a CD3 (e.g., CD3-zeta) intracellular domain. In certainembodiments, the intracellular signaling domain comprises a chimericCD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3zeta intracellular domain.

In certain embodiments, the CAR encompasses one or more, e.g., two ormore, costimulatory domains and an activation domain, e.g., primaryactivation domain, in the cytoplasmic portion. Exemplary CARs includeintracellular components of CD3-zeta, CD28, and 4-1BB.

In certain embodiments, the recombinant receptor further includes amarker and/or cells expressing the CAR or other antigen receptor furtherincludes a surrogate marker, such as a cell surface marker, which may beused to confirm transduction or engineering of the cell to express thereceptor. In certain embodiments, the marker includes all or part (e.g.,truncated form) of CD34, a NGFR, or epidermal growth factor receptor,such as truncated version of such a cell surface receptor (e.g., tEGFR).In certain embodiments, the nucleic acid encoding the marker is operablylinked to a polynucleotide encoding for a linker sequence, such as acleavable linker sequence, e.g., T2A. For example, a marker, andoptionally a linker sequence, can be any as disclosed in publishedpatent application No. WO2014031687. For example, the marker can be atruncated EGFR (EGFRt) that is, optionally, linked to a linker sequence,such as a T2A cleavable linker sequence.

In certain embodiments, the marker is a molecule, e.g., cell surfaceprotein, not naturally found on T cells or not naturally found on thesurface of T cells, or a portion thereof. In certain embodiments, themolecule is a non-self molecule, e.g., non-self protein, i.e., one thatis not recognized as “self” by the immune system of the host into whichthe cells will be adoptively transferred.

In certain embodiments, the marker serves no therapeutic function and/orproduces no effect other than to be used as a marker for geneticengineering, e.g., for selecting cells successfully engineered. In otherembodiments, the marker may be a therapeutic molecule or moleculeotherwise exerting some desired effect, such as a ligand for a cell tobe encountered in vivo, such as a costimulatory or immune checkpointmolecule to enhance and/or dampen responses of the cells upon adoptivetransfer and encounter with ligand.

In certain embodiments, CARs are referred to as first, second, and/orthird generation CARs. In certain embodiments, a first generation CAR isone that solely provides a CD3-chain induced signal upon antigenbinding, e.g., does not comprise a costimulatory signaling region. Incertain embodiments, a second-generation CARs is one that provides sucha signal and costimulatory signal, such as a CAR including acostimulatory signaling region, e.g., an intracellular signaling domainor a portion thereof of a costimulatory molecule, such as CD28 or 4-1BB.In certain embodiments, a third generation CAR is one that includesmultiple costimulatory regions, e.g., intracellular signaling domains ofdifferent costimulatory molecules (e.g., CD28 and 4-1BB).

In certain embodiments, the CAR includes an antibody, e.g., an antibodyfragment, a transmembrane domain that is or includes a transmembraneportion of CD28 or a functional variant thereof, and an intracellularsignaling domain comprising a signaling portion of CD28 or a functionalvariant thereof and a signaling portion of CD3 zeta or afunctionalvariant thereof. In certain embodiments, the CAR includes an antibody,e.g., an antibody fragment, a transmembrane domain that is or includes atransmembrane portion of CD28 or a functional variant thereof, and anintracellular signaling domain comprising a signaling portion of a 4-1BBor a functional variant thereof and a signaling portion of CD3 zeta or afunctional variant thereof. In certain embodiments, the CAR includes anantibody, e.g., an antibody fragment, a transmembrane domain that is orincludes a transmembrane portion of CD8 or a functional variant thereof,and an intracellular signaling domain comprising a signaling portion ofa 4-1BB or a functional variant thereof and a signaling portion of CD3zeta or a functional variant thereof. In certain embodiments, thereceptor further includes a spacer comprising a portion of an Igmolecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4hinge, such as a hinge-only spacer.

In certain embodiments, the CAR includes an antibody such as an antibodyfragment, including scFvs, a spacer, such as a spacer containing aportion of an immunoglobulin molecule, such as a hinge region and/or oneor more constant regions of a heavy chain molecule, such as an Ig-hingecontaining spacer, a transmembrane domain containing all or a portion ofa CD28-derived transmembrane domain, a CD28-derived intracellularsignaling domain, and a CD3 zeta signaling domain. In certainembodiments, the CAR includes an antibody or a fragment thereof, such asscFv, a spacer such as any of the Ig-hinge containing spacers, aCD28-derived transmembrane domain, a 4-1BB-derived intracellularsignaling domain, and a CD3 zeta-derived signaling domain. In certainembodiments, the CAR includes an antibody or a fragment thereof, such asscFv, a spacer such as any of the Ig-hinge containing spacers, aCD8-derived transmembrane domain, a 4-1BB-derived intracellularsignaling domain, and a CD3 zeta-derived signaling domain.

In certain embodiments, a nucleic acid molecule encoding such CARconstruct further includes a sequence encoding a T2A ribosomal skipelement and/or a tEGFR sequence, e.g., downstream of the sequenceencoding the CAR. In certain embodiments, T cells expressing arecombinant receptor (e.g. CAR) can also be generated to express atruncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g. byintroduction of a construct encoding the CAR and EGFRt separated by aT2A ribosome switch to express two proteins from the same construct),which then can be used as a marker to detect such cells (see e.g. U.S.Pat. No. 8,802,374).

The recombinant receptors, such as CARs, expressed by the cellsadministered to the subject generally recognize or specifically bind toa molecule that is expressed in, associated with, and/or specific forthe disease or condition or cells thereof being treated. Upon specificbinding to the molecule, e.g., antigen, the receptor generally deliversan immunostimulatory signal, such as an ITAM-transduced signal, into thecell, thereby promoting an immune response targeted to the disease orcondition. For example, In certain embodiments, the cells express a CARthat specifically binds to an antigen expressed by a cell or tissue ofthe disease or condition or associated with the disease or condition.

7.3.2. TCRS

In certain embodiments, the recombinant receptors include recombinant Tcell receptors (TCRs) and/or TCRs cloned from naturally occurring Tcells. In certain embodiments, a high-affinity T cell clone for a targetantigen (e.g., a cancer antigen) is identified, isolated from a patient,and introduced into the cells. In certain embodiments, the TCR clone fora target antigen has been generated in transgenic mice engineered withhuman immune system genes (e.g., the human leukocyte antigen system, orHLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) ClinCancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808.In certain embodiments, phage display is used to isolate TCRs against atarget antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med.14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354.

In certain embodiments, after the T-cell clone is obtained, the TCRalpha and beta chains are isolated and cloned into a gene expressionvector. In certain embodiments, the TCR alpha and beta genes are linkedvia a picornavirus 2A ribosomal skip peptide so that both chains arecoexpression. In certain embodiments, genetic transfer of the TCR isaccomplished via retroviral or lentiviral vectors, or via transposons(see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of theAmerican Society of Gene Therapy. 13:1050-1063; Frecha et al. (2010)Molecular Therapy: The Journal of the American Society of Gene Therapy.18:1748-1757; an Hackett et al. (2010) Molecular Therapy: The Journal ofthe American Society of Gene Therapy. 18:674-683.

7.4. GENETICALLY ENGINEERED CELLS AND METHODS OF PRODUCING CELLS

In certain embodiments, the provided methods include administering to asubject having a disease or condition cells expressing a recombinantreceptor. Various methods for the introduction of genetically engineeredcomponents, e.g., recombinant receptors, e.g., CARs or TCRs, are wellknown and may be used with the provided methods and compositions.Exemplary methods include those for transfer of nucleic acids encodingthe receptors, including via viral, e.g., retroviral or lentiviral,transduction, transposons, and electroporation.

Among the cells expressing the receptors and administered by theprovided methods are engineered cells. The genetic engineering generallyinvolves introduction of a nucleic acid encoding the recombinant orengineered component into a composition containing the cells, such as byretroviral transduction, transfection, or transformation.

7.4.1. VECTORS AND METHODS FOR GENETIC ENGINEERING

In certain embodiments, the recombinant nucleic acids are transferredinto cells using recombinant infectious virus particles, such as, e.g.,vectors derived from simian virus 40 (SV40), adenoviruses,adeno-associated virus (AAV). In certain embodiments, the recombinantnucleic acids are transferred into T cells using recombinant lentiviralvectors or retroviral vectors, such as gamma-retroviral vectors (see,e.g., Koste et al. (2014) Gene Therapy 2014 Apr. 3. doi:10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46;Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al.,Trends Biotechnol. 2011 Nov. 29(11): 550-557.

In certain embodiments, the retroviral vector has a long terminal repeatsequence (LTR), e.g., a retroviral vector derived from the Moloneymurine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV),murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV),spleen focus forming virus (SFFV), or adeno-associated virus (AAV). Mostretroviral vectors are derived from murine retroviruses. In certainembodiments, the retroviruses include those derived from any avian ormammalian cell source. The retroviruses typically are amphotropic,meaning that they are capable of infecting host cells of severalspecies, including humans. In one embodiment, the gene to be expressedreplaces the retroviral gag, pol and/or env sequences. A number ofillustrative retroviral systems have been described (e.g., U.S. Pat.Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989)BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14;Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc.Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993)Cur. Opin. Genet. Develop. 3:102-109.

Methods of lentiviral transduction are known. Exemplary methods aredescribed in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701;Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009)Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood.102(2): 497-505.

In certain embodiments, the recombinant nucleic acids are transferredinto the cells via electroporation (see, e.g., Chicaybam et al, (2013)PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16):1431-1437). In certain embodiments, recombinant nucleic acids aretransferred into T cells via transposition (see, e.g., Manuri et al.(2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec TherNucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic materialin immune cells include calcium phosphate transfection (e.g., asdescribed in Current Protocols in Molecular Biology, John Wiley & Sons,New York. N.Y.), protoplast fusion, cationic liposome-mediatedtransfection; tungsten particle-facilitated microparticle bombardment(Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNAco-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

Other approaches and vectors for transfer of the nucleic acids encodingthe recombinant products are those described, e.g., in internationalpatent application, Publication No.: WO2014055668, and U.S. Pat. No.7,446,190.

In certain embodiments, the cells (e.g., T cells) may be transfectedeither during or after expansion e.g. with a T cell receptor (TCR) or achimeric antigen receptor (CAR). This transfection for the introductionof the gene of the desired receptor can be carried out with any suitableretroviral vector, for example. The genetically modified cell populationcan then be liberated from the initial stimulus (the CD3/CD28 stimulus,for example) and subsequently be stimulated with a second type ofstimulus e.g. via a de novo introduced receptor). This second type ofstimulus may include an antigenic stimulus in form of a peptide/WICmolecule, the cognate (cross-linking) ligand of the geneticallyintroduced receptor (e.g. natural ligand of a CAR) or any ligand (suchas an antibody) that directly binds within the framework of the newreceptor (e.g. by recognizing constant regions within the receptor).See, for example, Cheadle et al, “Chimeric antigen receptors for T-cellbased therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al.,Chimeric Antigen Receptor Therapy for Cancer Annual Review of MedicineVol. 65: 333-347 (2014).

In some cases, a vector may be used that does not require that the cells(e.g., T cells) are activated. In some such instances, the cells may beselected and/or transduced prior to activation. Thus, the cells may beengineered prior to, or subsequent to culturing of the cells, and insome cases at the same time as or during at least a portion of theculturing.

Among additional nucleic acids, e.g., genes for introduction are thoseto improve the efficacy of therapy, such as by promoting viabilityand/or function of transferred cells; genes to provide a genetic markerfor selection and/or evaluation of the cells, such as to assess in vivosurvival or localization; genes to improve safety, for example, bymaking the cell susceptible to negative selection in vivo as describedby Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell etal., Human Gene Therapy 3:319-338 (1992); see also the publications ofPCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use ofbifunctional selectable fusion genes derived from fusing a dominantpositive selectable marker with a negative selectable marker. See, e.g.,Riddell et al., U.S. Pat. No. 6,040,177, at columns 14-17.

7.4.2. CELLS AND PREPARATION OF CELLS FOR GENETIC ENGINEERING

In certain embodiments, the nucleic acids are heterologous, i.e.,normally not present in a cell or sample obtained from the cell, such asone obtained from another organism or cell, which for example, is notordinarily found in the cell being engineered and/or an organism fromwhich such cell is derived. In certain embodiments, the nucleic acidsare not naturally occurring, such as a nucleic acid not found in nature,including one comprising chimeric combinations of nucleic acids encodingvarious domains from multiple different cell types.

The cells generally are eukaryotic cells, such as mammalian cells, andtypically are human cells. In certain embodiments, the cells are derivedfrom the blood, bone marrow, lymph, or lymphoid organs, are cells of theimmune system, such as cells of the innate or adaptive immunity. Incertain embodiments, the cell is a cell of the lymphoid lineage or acell of the myeloid lineage. In certain embodiments, the cell is a cellof the lymphoid lineage. In certain embodiments, the cell of thelymphoid lineage is selected from the group consisting of a T cell, aNatural Killer (NK) cell, a stem cell from which lymphoid cells may bedifferentiated. Stem cells can be multipotent and pluripotent stemcells. In certain embodiments, the stem cell is a pluripotent stem cell.In certain embodiments, the pluripotent stem cell is an embryoid stemcell or an induced pluripotent stem cell. The cells typically areprimary cells, such as those isolated directly from a subject and/orisolated from a subject and frozen. In certain embodiments, the cellsinclude one or more subsets of T cells or other cell types, such aswhole T cell populations, CD4⁺ cells, CD8⁺ cells, and subpopulationsthereof, such as those defined by function, activation state, maturity,potential for differentiation, expansion, recirculation, localization,and/or persistence capacities, antigen-specificity, type of antigenreceptor, presence in a particular organ or compartment, marker orcytokine secretion profile, and/or degree of differentiation. Withreference to the subject to be treated, the cells may be allogeneicand/or autologous. Among the methods include off-the-shelf methods. Incertain embodiments, such as for off-the-shelf technologies, the cellsare pluripotent and/or multipotent, such as stem cells, such as inducedpluripotent stem cells (iPSCs). In certain embodiments, the methodsinclude isolating cells from the subject, preparing, processing,culturing, and/or engineering them, and re-introducing them into thesame subject, before or after cryopreservation.

In certain embodiments, the cells are T cells. Non-limiting examples ofsub-types and subpopulations of T cells and/or of CD4⁺ and/or of CD8⁺ Tcells include naïve T (TN) cells, Natural Killer T cells, effector Tcells (T_(EFF)), memory T cells and sub-types thereof, such as stem cellmemory T (T_(SCM)), central memory T (T_(CM)), effector memory T(T_(EM)), terminally differentiated effector memory T cells,tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells,helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT)cells, regulatory T (Treg) cells, helper T cells (e.g., TH1 cells, TH2cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, and follicularhelper T cells), alpha/beta T cells, and delta/gamma T cells.

In certain embodiments, the cells are natural killer (NK) cells. Incertain embodiments, the cells are monocytes or granulocytes, e.g.,myeloid cells, macrophages, neutrophils, dendritic cells, mast cells,eosinophils, and/or basophils.

In certain embodiments, the cells are cells of the myeloid lineage.Non-limiting examples of cells of the myeloid lineage include monocytes,macrophages, neutrophils, dendritic cells, basophils, neutrophils,eosinophils, megakaryocytes, mast cell, erythrocyte, thrombocytes, andstem cells from which myeloid cells may be differentiated. In certainembodiments, the stem cell is a pluripotent stem cell (e.g., anembryonic stem cell or an induced pluripotent stem cell).

In certain embodiments, the cells include one or more nucleic acidmolecules introduced via genetic engineering, and thereby expressrecombinant or genetically engineered products of such nucleic acids. Incertain embodiments, the nucleic acid molecules are heterologous, i.e.,normally not present in a cell or sample obtained from the cell, such asone obtained from another organism or cell, which for example, is notordinarily found in the cell being engineered and/or an organism fromwhich such cell is derived. In certain embodiments, the nucleic acidmolecules are not naturally occurring, such as a nucleic acid moleculenot found in nature, including one comprising chimeric combinations ofnucleic acid molecules encoding various domains from multiple differentcell types.

In certain embodiments, preparation of the engineered cells includes oneor more culture and/or preparation steps. The cells for introduction ofthe nucleic acid molecule encoding the transgenic receptor such as theCAR, may be isolated from a sample, such as a biological sample, e.g.,one obtained from or derived from a subject. In certain embodiments, thesubject from which the cell is isolated is one having the disease orcondition or in need of a cell therapy or to which cell therapy will beadministered. In certain embodiments, the subject is a human in need ofa particular therapeutic intervention, such as the adoptive cell therapyfor which cells are being isolated, processed, and/or engineered.

Accordingly, the cells in certain embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In certain embodiments, the sample from which the cells are derived orisolated is blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In certain embodiments, the cells are derived from cell lines, e.g., Tcell lines. In certain embodiments, the cells are obtained from axenogeneic source, for example, from mouse, rat, non-human primate, andpig.

In certain embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In certainembodiments, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In certain embodiments, cells areseparated based on one or more property, such as density, adherentproperties, size, sensitivity and/or resistance to particularcomponents.

In certain embodiments, cells from the circulating blood of a subjectare obtained, e.g., by apheresis or leukapheresis. The samples, incertain embodiments, comprise lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in certain embodiments includes cells otherthan red blood cells and platelets.

In certain embodiments, the blood cells collected from the subject arewashed, e.g., to remove the plasma fraction and to place the cells in anappropriate buffer or media for subsequent processing steps. In certainembodiments, the cells are washed with phosphate buffered saline (PBS).In certain embodiments, the wash solution lacks calcium and/or magnesiumand/or many or all divalent cations. In certain embodiments, a washingstep is accomplished a semi-automated “flow-through” centrifuge (forexample, the Cobe 2991 cell processor, Baxter) according to themanufacturer's instructions. In certain embodiments, a washing step isaccomplished by tangential flow filtration (TFF) according to themanufacturer's instructions. In certain embodiments, the cells areresuspended in a variety of biocompatible buffers after washing, suchas, for example, Ca⁺⁺/Mg⁺⁺ free PBS. In certain embodiments, componentsof a blood cell sample are removed and the cells directly resuspended inculture media.

In certain embodiments, the methods include density-based cellseparation methods, such as the preparation of white blood cells fromperipheral blood by lysing the red blood cells and centrifugationthrough a Percoll or Ficoll gradient.

In certain embodiments, the isolation methods include separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In certainembodiments, any known method for separation based on such markers maybe used. In certain embodiments, the separation is affinity- orimmunoaffinity-based separation. For example, the isolation in certainembodiments includes separation of cells and cell populations based onthe cells' expression or expression level of one or more markers,typically cell surface markers, for example, by incubation with anantibody or binding partner that specifically binds to such markers,followed generally by washing steps and separation of cells having boundthe antibody or binding partner, from those cells having not bound tothe antibody or binding partner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In certain embodiments, both fractionsare retained for further use. In certain embodiments, negative selectioncan be particularly useful where no antibody is available thatspecifically identifies a cell type in a heterogeneous population, suchthat separation is best carried out based on markers expressed by cellsother than the desired population.

The separation need not result in 100% enrichment or removal of aparticular cell population or cells expressing a particular marker. Forexample, positive selection of or enrichment for cells of a particulartype, such as those expressing a marker, refers to increasing the numberor percentage of such cells, but need not result in a complete absenceof cells not expressing the marker. Likewise, negative selection,removal, or depletion of cells of a particular type, such as thoseexpressing a marker, refers to decreasing the number or percentage ofsuch cells, but need not result in a complete removal of all such cells.

In certain embodiments, multiple rounds of separation steps are carriedout, where the positively or negatively selected fraction from one stepis subjected to another separation step, such as a subsequent positiveor negative selection. In certain embodiments, a single separation stepcan deplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

For example, in certain embodiments, specific subpopulations of T cells,such as cells positive or expressing detection (e.g., high) levels ofone or more surface markers, e.g., CD28⁺, CD62L⁺, CCR7⁺, CD27⁺, CD127⁺,CD4⁺, CD8⁺, CD45RA⁺, and/or CD45RO⁺ T cells, are isolated by positive ornegative selection techniques.

For example, CD3⁺, CD28⁺ T cells can be positively selected usingCD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 TCell Expander).

In certain embodiments, isolation is carried out by enrichment for aparticular cell population by positive selection, or depletion of aparticular cell population, by negative selection. In certainembodiments, positive or negative selection is accomplished byincubating cells with one or more antibodies or other binding agent thatspecifically bind to one or more surface markers expressed or expressed(marker⁺) at a relatively higher level (marker^(high)) on the positivelyor negatively selected cells, respectively.

In certain embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD14. In certainembodiments, a CD4⁺ or CD8⁺ selection step is used to separate CD4⁺helper and CD8⁺ cytotoxic T cells. Such CD4⁺ and CD8⁺ populations can befurther sorted into sub-populations by positive or negative selectionfor markers expressed or expressed to a relatively higher degree on oneor more naive, memory, and/or effector T cell subpopulations.

In certain embodiments, CD8⁺ cells are further enriched for or depletedof naive, central memory, effector memory, and/or central memory stemcells, such as by positive or negative selection based on surfaceantigens associated with the respective subpopulation. In certainembodiments, enrichment for central memory T (T_(CM)) cells is carriedout to increase efficacy, such as to improve long-term survival,expansion, and/or engraftment following administration, which in certainembodiments is particularly robust in such sub-populations. See Terakuraet al. (2012) Blood. 1:72-82; Wang et al. (2012) J Immunother.35(9):689-701. In certain embodiments, combining T_(CM)-enriched CD8⁺ Tcells and CD4⁺ T cells further enhances efficacy.

In certain embodiments, memory T cells are present in both CD62L⁺ andCD62L⁻ subsets of CD8⁺ peripheral blood lymphocytes. PBMC can beenriched for or depleted of CD62L⁻CD8⁺ and/or CD62L⁺CD8⁺ fractions, suchas using anti-CD8 and anti-CD62L antibodies.

In certain embodiments, the enrichment for central memory T (T_(CM))cells is based on positive or high surface expression of CD45RO, CD62L,CCR7, CD28, CD3, and/or CD 127; in certain embodiments, it is based onnegative selection for cells expressing or highly expressing CD45RAand/or granzyme B. In certain embodiments, isolation of a CD8⁺population enriched for T_(CM) cells is carried out by depletion ofcells expressing CD4, CD14, CD45RA, and positive selection or enrichmentfor cells expressing CD62L. In one aspect, enrichment for central memoryT (T_(CM)) cells is carried out starting with a negative fraction ofcells selected based on CD4 expression, which is subjected to a negativeselection based on expression of CD14 and CD45RA, and a positiveselection based on CD62L. Such selections in certain embodiments arecarried out simultaneously and in other aspects are carried outsequentially, in either order. In certain embodiments, the same CD4expression-based selection step used in preparing the CD8⁺ cellpopulation or subpopulation, also is used to generate the CD4⁺ cellpopulation or sub-population, such that both the positive and negativefractions from the CD4-based separation are retained and used insubsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

In certain embodiments, a sample of PBMCs or other white blood cellsample is subjected to selection of CD4⁺ cells, where both the negativeand positive fractions are retained. The negative fraction then issubjected to negative selection based on expression of CD14 and CD45RAor CD19, and positive selection based on a marker characteristic ofcentral memory T cells, such as CD62L or CCR7, where the positive andnegative selections are carried out in either order.

CD4⁺ T helper cells are sorted into naïve, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4⁺ lymphocytes can be obtained by standard methods. In certainembodiments, naive CD4⁺ T lymphocytes are CD45RO⁻, CD45RA⁺, CD62L⁺, CD4⁺T cells. In certain embodiments, central memory CD4⁺ cells are CD62L⁺and CD45RO⁺. In certain embodiments, effector CD4⁺ cells are CD62L⁻ andCD45RO⁻.

In certain embodiments, to enrich for CD4⁺ cells by negative selection,a monoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, the antibodyor binding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection. For example, in certain embodiments,the cells and cell populations are separated or isolated usingimmunomagnetic (or affinity magnetic) separation techniques (reviewed inMethods in Molecular Medicine, vol. 58: Metastasis Research Protocols,Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A.Brooks and U. Schumacher© Humana Press Inc., Totowa, N.J.).

In certain embodiments, the preparation methods include steps forfreezing, e.g., cryopreserving, the cells, either before or afterisolation, incubation, and/or engineering. In certain embodiments, thefreeze and subsequent thaw step removes granulocytes and, to someextent, monocytes in the cell population. In certain embodiments, thecells are suspended in a freezing solution, e.g., following a washingstep to remove plasma and platelets. Any of a variety of known freezingsolutions and parameters in certain embodiments may be used. One exampleinvolves using PBS containing 20% DMSO and 8% human serum albumin (HSA),or other suitable cell freezing media. This is then diluted 1:1 withmedia so that the final concentration of DMSO and HSA are 10% and 4%,respectively. The cells are generally then frozen to −80° C. at a rateof 1° per minute and stored in the vapor phase of a liquid nitrogenstorage tank.

In certain embodiments, the cells are incubated and/or cultured prior toor in connection with genetic engineering. The incubation steps caninclude culture, cultivation, stimulation, activation, and/orpropagation. The incubation and/or engineering may be carried out in aculture vessel, such as a unit, chamber, well, column, tube, tubing set,valve, vial, culture dish, bag, or other container for culture orcultivating cells. In certain embodiments, the compositions or cells areincubated in the presence of stimulating conditions or a stimulatoryagent. Such conditions include those designed to induce proliferation,expansion, activation, and/or survival of cells in the population, tomimic antigen exposure, and/or to prime the cells for geneticengineering, such as for the introduction of a recombinant antigenreceptor.

The conditions can include one or more of particular media, temperature,oxygen content, carbon dioxide content, time, agents, e.g., nutrients,amino acids, antibiotics, ions, and/or stimulatory factors, such ascytokines, chemokines, antigens, binding partners, fusion proteins,recombinant soluble receptors, and any other agents designed to activatethe cells.

In certain embodiments, the stimulating conditions or agents include oneor more agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In certain embodiments,the agent turns on or initiates TCR/CD3 intracellular signaling cascadein a T cell. Such agents can include antibodies, such as those specificfor a TCR component and/or costimulatory receptor, e.g., anti-CD3,anti-CD28, for example, bound to solid support such as a bead, and/orone or more cytokines. Optionally, the expansion method may furthercomprise the step of adding anti-CD3 and/or anti CD28 antibody to theculture medium (e.g., at a concentration of at least about 0.5 ng/ml).In certain embodiments, the stimulating agents include IL-2 and/orIL-15, for example, an IL-2 concentration of at least about 10 units/mL.

In certain embodiments, incubation is carried out in accordance withtechniques such as those described in U.S. Pat. No. 6,040,177 to Riddellet al., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakuraet al. (2012) Blood. 1:72-82, and/or Wang et al. (2012) J Immunother.35(9):689-701.

In certain embodiments, the T cells are expanded by adding to aculture-initiating composition feeder cells, such as non-dividingperipheral blood mononuclear cells (PBMC), (e.g., such that theresulting population of cells includes at least about 5, 10, 20, or 40or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T cells). In certain embodiments,the non-dividing feeder cells can comprise gamma-irradiated PBMC feedercells. In certain embodiments, the PBMC are irradiated with gamma raysin the range of about 3000 to 3600 rads to prevent cell division. Incertain embodiments, the feeder cells are added to culture medium priorto the addition of the populations of T cells.

In certain embodiments, the stimulating conditions include temperaturesuitable for the growth of human T lymphocytes, for example, at leastabout 25 degrees Celsius, generally at least about 30 degrees, andgenerally at about 37 degrees Celsius. Optionally, the incubation mayfurther comprise adding non-dividing EBV-transformed lymphoblastoidcells (LCL) as feeder cells. LCL can be irradiated with gamma rays inthe range of about 6000 to 10,000 rads. The LCL feeder cells in certainembodiments is provided in any suitable amount, such as a ratio of LCLfeeder cells to initial T lymphocytes of at least about 10:1.

In embodiments, antigen-specific T cells, such as antigen-specific CD4⁺and/or CD8⁺ T cells, are obtained by stimulating naive or antigenspecific T lymphocytes with antigen. For example, antigen-specific Tcell lines or clones can be generated to cytomegalovirus antigens byisolating T cells from infected subjects and stimulating the cells invitro with the same antigen.

7.5. COMPOSITIONS AND FORMULATIONS OF GENETICALLY ENGINEERED CELLS

In certain embodiments, the cells engineered with a recombinantreceptor, e.g. CAR or TCR, is provided as a composition or formulation,such as a pharmaceutical composition or formulation. Such compositionscan be used in accord with the provided methods, such as in theprevention or treatment of diseases, conditions, and disorders, or indetection, diagnostic, and prognostic methods. The term “pharmaceuticalformulation” refers to a preparation which is in such form as to permitthe biological activity of an active ingredient contained therein to beeffective, and which includes no additional components which areunacceptably toxic to a subject to which the formulation would beadministered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

In certain embodiments, the choice of carrier is determined in part bythe particular cell or agent and/or by the method of administration.Accordingly, there are a variety of suitable formulations. For example,the pharmaceutical composition can contain preservatives. Suitablepreservatives may include, for example, methylparaben, propylparaben,sodium benzoate, and benzalkonium chloride. In certain embodiments, amixture of two or more preservatives is used. The preservative ormixtures thereof are typically present in an amount of about 0.0001% toabout 2% by weight of the total composition. Carriers are described,e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.(1980). Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG).

Buffering agents in certain embodiments are included in thecompositions. Suitable buffering agents include, for example, citricacid, sodium citrate, phosphoric acid, potassium phosphate, and variousother acids and salts. In certain embodiments, a mixture of two or morebuffering agents is used. The buffering agent or mixtures thereof aretypically present in an amount of about 0.001% to about 4% by weight ofthe total composition. Methods for preparing administrablepharmaceutical compositions are known. Exemplary methods are describedin more detail in, for example, Remington: The Science and Practice ofPharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulation or composition may also contain more than one activeingredient useful for the particular indication, disease, or conditionbeing prevented or treated with the cells or agents, where therespective activities do not adversely affect one another. Such activeingredients are suitably present in combination in amounts that areeffective for the purpose intended. Thus, in certain embodiments, thepharmaceutical composition further includes other pharmaceuticallyactive agents or drugs, such as chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, vincristine, etc. In certainembodiments, the agents or cells are administered in the form of a salt,e.g., a pharmaceutically acceptable salt. Suitable pharmaceuticallyacceptable acid addition salts include those derived from mineral acids,such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric,and sulphuric acids, and organic acids, such as tartaric, acetic,citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic,and arylsulphonic acids, for example, p-toluenesulphonic acid.

Active ingredients may be entrapped in microcapsules, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.In certain embodiments, the pharmaceutical composition is formulated asan inclusion complex, such as cyclodextrin inclusion complex, or as aliposome. Liposomes can serve to target the agent or host cells (e.g.,T-cells or NK cells) to a particular tissue. Many methods are availablefor preparing liposomes, such as those described in, for example, Szokaet al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Pat. Nos.4,235,871, 4,501,728, 4,837,028, and 5,019,369.

The pharmaceutical composition in certain embodiments can employtime-released, delayed release, and sustained release delivery systemssuch that the delivery of the composition occurs prior to, and withsufficient time to cause, sensitization of the site to be treated. Manytypes of release delivery systems are available and known. Such systemscan avoid repeated administrations of the composition, therebyincreasing convenience to the subject and the physician.

In certain embodiments, the pharmaceutical composition includes agentsor cells in amounts effective to treat or prevent the disease orcondition, such as a therapeutically effective or prophylacticallyeffective amount. In certain embodiments, therapeutic or prophylacticefficacy is monitored by periodic assessment of treated subjects. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is repeated until a desired suppression ofdisease symptoms occurs. However, other dosage regimens may be usefuland can be determined. The desired dosage can be delivered by a singlebolus administration of the composition, by multiple bolusadministrations of the composition, or by continuous infusionadministration of the composition.

Methods for administration of cells for adoptive cell therapy are knownand may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

In certain embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by autologous transfer, inwhich the cells are isolated and/or otherwise prepared from the subjectwho is to receive the cell therapy, or from a sample derived from such asubject. Thus, in certain embodiments, the cells are derived from asubject, e.g., patient, in need of a treatment and the cells, andfollowing isolation and processing are administered to the same subject.

In certain embodiments, the cell therapy, e.g., adoptive cell therapy,e.g., adoptive T cell therapy, is carried out by allogeneic transfer, inwhich the cells are isolated and/or otherwise prepared from a subjectother than a subject who is to receive or who ultimately receives thecell therapy, e.g., a first subject. In such embodiments, the cells thenare administered to a different subject, e.g., a second subject, of thesame species. In certain embodiments, the first and second subjects aregenetically identical. In certain embodiments, the first and secondsubjects are genetically similar. In certain embodiments, the secondsubject expresses the same HLA class or supertype as the first subject.

The agents or cells can be administered by any suitable means, forexample, by bolus infusion, by injection, e.g., intravenous orsubcutaneous injections, intraocular injection, periocular injection,subretinal injection, intravitreal injection, trans-septal injection,subscleral injection, intrachoroidal injection, intracameral injection,subconjectval injection, subconjuntival injection, sub-Tenon'sinjection, retrobulbar injection, peribulbar injection, or posteriorjuxtascleral delivery. In certain embodiments, they are administered byparenteral, intrapulmonary, and intranasal, and, if desired for localtreatment, intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In certain embodiments, a given dose isadministered by a single bolus administration of the cells or agent. Incertain embodiments, it is administered by multiple bolusadministrations of the cells or agent, for example, over a period of nomore than 3 days, or by continuous infusion administration of the cellsor agent.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of agent oragents, the type of cells or recombinant receptors, the severity andcourse of the disease, whether the agent or cells are administered forpreventive or therapeutic purposes, previous therapy, the subject'sclinical history and response to the agent or the cells, and thediscretion of the attending physician. In certain embodiments, thecompositions are suitably administered to the subject at one time orover a series of treatments.

The cells or agents may be administered using standard administrationtechniques, formulations, and/or devices. Provided are formulations anddevices, such as syringes and vials, for storage and administration ofthe compositions. With respect to cells, administration can beautologous or heterologous. For example, cells or progenitors can beobtained from one subject, and administered to the same subject or adifferent, compatible subject. Peripheral blood derived cells or theirprogeny (e.g., in vivo, ex vivo or in vitro derived) can be administeredvia localized injection, including catheter administration, systemicinjection, localized injection, intravenous injection, or parenteraladministration. When administering a therapeutic composition (e.g., apharmaceutical composition containing a genetically modified cell or anagent that treats or ameliorates symptoms of neurotoxicity), it willgenerally be formulated in a unit dosage injectable form (solution,suspension, emulsion).

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In certain embodiments, theagent or cell populations are administered parenterally. The term“parenteral,” as used herein, includes intravenous, intramuscular,subcutaneous, rectal, vaginal, and intraperitoneal administration. Incertain embodiments, the agent or cell populations are administered to asubject using peripheral systemic delivery by intravenous,intraperitoneal, or subcutaneous injection.

In certain embodiments, compositions are provided as sterile liquidpreparations, e.g., isotonic aqueous solutions, suspensions, emulsions,dispersions, or viscous compositions, which may in certain embodimentsbe buffered to a selected pH. Liquid preparations are normally easier toprepare than gels, other viscous compositions, and solid compositions.Additionally, liquid compositions are somewhat more convenient toadminister, especially by injection. Viscous compositions, on the otherhand, can be formulated within the appropriate viscosity range toprovide longer contact periods with specific tissues. Liquid or viscouscompositions can comprise carriers, which can be a solvent or dispersingmedium containing, for example, water, saline, phosphate bufferedsaline, polyoi (for example, glycerol, propylene glycol, liquidpolyethylene glycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the agentor cells in a solvent, such as in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose, dextrose, or the like. The compositions can also belyophilized. The compositions can contain auxiliary substances such aswetting, dispersing, or emulsifying agents (e.g., methylcellulose), pHbuffering agents, gelling or viscosity enhancing additives,preservatives, flavoring agents, colors, and the like, depending uponthe route of administration and the preparation desired. Standard textsmay in certain embodiments be consulted to prepare suitablepreparations.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Prolonged absorption of the injectable pharmaceutical form canbe brought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

7.6. DOSING OF THE GENETICALLY ENGINEERED CELLS

In certain embodiments, the size or timing of the doses of thegenetically engineered cells is determined as a function of theparticular disease or condition in the subject. It is within the levelof a skilled artisan to empirically determine the size or timing of thedoses for a particular disease in view of the provided description. Forthe prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of cells orrecombinant receptors, the severity and course of the disease, whetherthe cells are administered for preventive or therapeutic purposes,previous therapy, the subject's clinical history and response to thecells, and the discretion of the attending physician. In certainembodiments, the compositions and cells are suitably administered to thesubject at one time or over a series of treatments.

In the context of adoptive cell therapy, administration of a given“dose” referring to the cells encompasses administration of the givenamount or number of cells as a single composition and/or singleuninterrupted administration, e.g., as a single injection or continuousinfusion, and also encompasses administration of the given amount ornumber of cells as a split dose, provided in multiple individualcompositions or infusions, over a specified period of time, which is nomore than 3 days. Thus, in some contexts, the first or consecutive doseis a single or continuous administration of the specified number ofcells, given or initiated at a single point in time. In some contexts,however, the first or consecutive dose is administered in multipleinjections or infusions over a period of no more than three days, suchas once a day for three days or for two days or by multiple infusionsover a single day period.

Thus, in certain embodiments, the cells of the first dose areadministered in a single pharmaceutical composition. In certainembodiments, the cells of the consecutive dose are administered in asingle pharmaceutical composition.

In certain embodiments, the cells of the first dose are administered ina plurality of compositions, collectively containing the cells of thefirst dose. In certain embodiments, the cells of the consecutive doseare administered in a plurality of compositions, collectively containingthe cells of the consecutive dose. In certain embodiments, additionalconsecutive doses may be administered in a plurality of compositionsover a period of no more than 3 days.

The quantity of cells to be administered will vary for the subject beingtreated. In certain embodiments, between about 1×10⁴ and about 1×10¹⁰,between about 1×10⁵ and about 1×10⁹, or between about 1×10⁶ and about1×10⁸ of the genetically engineered cells (recombinantreceptor-expressing cells) are administered to a subject (e.g., a humansubject). More effective cells may be administered in even smallernumbers. In certain embodiments, at least about 1×10⁶, at least about1×10⁷, at least about 1×10⁸ (e.g., about 2×10⁸, about 3×10⁸, about4×10⁸, or about 5×10⁸) of the genetically engineered cells (recombinantreceptor-expressing cells) are administered to a subject (e.g., a humansubject). The precise determination of what would be considered aneffective dose may be based on factors individual to each subject,including their size, age, sex, weight, and condition of the particularsubject. Dosages can be readily ascertained by those skilled in the artfrom this disclosure and the knowledge in the art.

In certain embodiments, the numbers and/or concentrations of cells referto the number of recombinant receptor (e.g., CAR)-expressing cells. Incertain embodiments, the numbers and/or concentrations of cells refer tothe number or concentration of all cells, T cells, or peripheral bloodmononuclear cells (PBMCs) administered.

In certain embodiments, the size of the dose is determined based on oneor more criteria such as response of the subject to prior treatment,e.g. chemotherapy, disease burden in the subject, such as tumor load,bulk, size, or degree, extent, or type of metastasis, stage, and/orlikelihood or incidence of the subject developing toxic outcomes, e.g.,CRS, macrophage activation syndrome, tumor lysis syndrome,neurotoxicity, and/or a host immune response against the cells and/orrecombinant receptors being administered.

In certain embodiments, the size of the dose is determined by the burdenof the disease or condition in the subject. For example, in certainembodiments, the number of cells administered is determined based on thetumor burden that is present in the subject immediately prior toadministration of the dose of cells. In certain embodiments, the size ofthe dose is inversely correlated with disease burden. In certainembodiments, as in the context of a large disease burden, the subject isadministered a low number of cells.

In reference to cell numbers, in certain embodiments, such values referto numbers of recombinant receptor-expressing (e.g. CAR-expressing)cells; in other embodiments, they refer to number of T cells or PBMCs ortotal cells administered.

In certain embodiments, one or more further consecutive doses can beadministered. In certain embodiments, the number of cells administeredin the consecutive dose is the same as or similar to the number of cellsadministered in the first dose in any of the embodiments herein. Incertain embodiments, the particular dosage regimen is chosen to reduceor minimize toxicity in the subject upon administration of the cellsexpressing the recombinant receptor, such as described in InternationalPatent Application Publication No. WO2016/064929, which is incorporatedby reference herein.

In certain embodiments, if desired or need in a particular disease orcontext, the provided methods involve a consecutive dose of cellsadministered at an increased number, and hence at a higher dose, thanthe first dose of cells. In certain embodiments, methods of firstadministering a low dose of recombinant receptor-expressing (e.g.,CAR-expressing) cells can reduce the disease burden in subjects, such asfrom morphological disease status to minimal disease, so that subsequentadministration of a higher dose of recombinant receptor-expressing(e.g., CAR-expressing) cells in subjects is less likely to cause toxicoutcomes in a majority of subjects treated.

In certain embodiments, a subject can be assessed for tumor burden afteradministration of the first dose and prior to administration of theconsecutive dose to confirm that tumor burden has been reduced comparedto tumor burden present prior to treatment with the first dose. Incertain embodiments, if assessment of the subject indicates tumor burdenis reduced and/or that the subject exhibits non-morphological disease,for example molecularly detectable disease and/or minimal disease, theprovided methods include administering a consecutive dose of recombinantreceptor-expressing (e.g., CAR-expressing) cells that is the same orless than the first or initial dose. In certain embodiments, ifassessment of the subject indicates tumor burden is not reduced and/orthe subject exhibits morphological disease, the provided methods includeadministering a consecutive dose of recombinant receptor-expressing(e.g., CAR-expressing) cells that is higher than the first or initialdose.

In certain embodiments, a consecutive dose of recombinantreceptor-expressing (e.g., CAR-expressing) cells is administered to thesubject at a time after administration of the first or initial dose ofcells in which it is likely that tumor burden of the subject has beenreduced by the first dose. In certain embodiments, it is not necessarythat the tumor burden actually be reduced in all subjects prior toadministration of the consecutive dose, but that tumor burden is reducedon average in subjects treated, such as based on clinical data, in whicha majority of subjects treated with such a first dose exhibit a reducedtumor burden, such as at least about 50%, about 60%, about 70%, about80%, about 90%, about 95% or more of subjects treated with the first orinitial dose exhibit a reduced tumor burden. Generally at a point intime after disease burden has been reduced by the first dose or islikely to have been reduced by the first dose, a consecutive dose isadministered to the subject, thereby further reducing and/or eliminatingdisease or a symptom or outcome thereof or preventing expansion orprogression thereof. The context of reduced disease burden at the timeof the consecutive administration in certain embodiments reduces thelikelihood of exhaustion of the transferred cells, thereby improvingefficacy. The consecutive dose may be the same, lower, or a higher doseas compared with the first dose. In certain embodiments, multipleconsecutive doses are administered after a first dose.

In certain embodiments, the consecutive dose of recombinantreceptor-expressing (e.g., CAR-expressing) cells is administered at adose that is higher than the first dose so that an increased number ofrecombinant receptor-expressing (e.g., CAR-expressing) cells isadministered to the subject by the consecutive dose. In certainembodiments, a higher dose of recombinant receptor-expressing (e.g.,CAR-expressing) cells is one that can promote an increased response orefficacy, such as improved or greater reduction in tumor burden and/oran improved or greater overall survival time of the subject compared tothat achieved by administration of a lower dose or number of cells. Incertain embodiments, because administration of the first dose of cellscan reduce tumor burden in the subject, administration of theconsecutive dose at a higher number of cells can avoid or minimize CRSand/or neurotoxicity in the subject after administration of theconsecutive dose that can otherwise occur in subjects with morphologicaldisease.

In certain embodiments, the consecutive dose is larger than the firstdose. For example, in certain embodiments, the consecutive dose includesmore than about 1×10⁶ cells (e.g., about or at least about 2×10⁶, about3×10⁶, about 5×10⁶, about 1×10⁷, about 1×10⁸, or about 1×10⁹) of therecombinant receptor (e.g. CAR)-expressing cells. In certainembodiments, the amount or size of the consecutive dose is sufficient toreduce disease burden or an indicator thereof, and/or one or moresymptoms of the disease or condition. In certain embodiments, the doseis of a size effective to improve survival of the subject, for example,to induce survival, relapse-free survival, or event-free survival of thesubject for at least 6 months, or at least about 1, about 2, about 3,about 4, or about 5 years. In certain embodiments, the number ofrecombinant receptor (e.g. CAR)-expressing cells (e.g., CAR-expressing Tcells) administered and/or number of such cells administered per bodyweight of the subject in the consecutive dose is at least about 2-fold,about 3-fold, about 5-fold, about 10-fold greater than the numberadministered in the first dose. In certain embodiments, disease burden,tumor size, tumor volume, tumor mass, and/or tumor load or bulk isreduced following the consecutive dose by at least about 50%, about 60%,about 70%, about 80%, or about 90% or more compared to that immediatelyprior to the administration of the first dose or of the consecutivedose.

In certain embodiments, the number of cells administered in theconsecutive dose is lower than the number of cells administered in thefirst dose.

In certain embodiments, multiple consecutive doses are administeredfollowing the first dose, such that an additional dose or doses areadministered following administration of the consecutive dose. Incertain embodiments, the number of cells administered to the subject inthe additional dose or doses (i.e., the third, fourth, fifth, and soforth) is the same as or similar to the first dose and/or consecutivedose. In certain embodiments, the additional dose or doses are largerthan prior doses.

In certain embodiments, the timing of the consecutive dose(s) inrelation to the first and/or one another is designed to reduce the riskof unwanted toxic outcomes and promote maximum efficacy. In certainembodiments, the consecutive dose, such as the same, lower or higherconsecutive dose, is administered at a time at which disease burdenremains reduced in the subject or reduced in subjects on average, suchas based on clinical data, but at which the risk of CRS and/orneurotoxicity remain low. In certain embodiments, a consecutive dose isgenerally given at a point in time relative to the first or previousdose at which the risk of a toxic outcome or symptom or biochemicalindicator thereof—such as CRS or neurotoxicity, macrophage activationsyndrome, or tumor lysis syndrome—is at or below an acceptable level.For example, the consecutive dose may be administered after a toxicoutcome has peaked and is declining or has declined below an acceptablelevel following the initial dose. In certain embodiments, theappropriate timing is determined by monitoring and/or assessing thepresence of one or more symptoms or outcomes associated with the toxicevent and delivering the consecutive dose after determining that thesymptom or outcome is at or below an acceptable level.

Once the cells are administered to the subject (e.g., human), thebiological activity of the engineered cells can be measured by any of anumber of known methods. Parameters to assess include specific bindingof an engineered or natural T cell or other immune cell to antigen, invivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. Incertain embodiments, the ability of the engineered cells to destroytarget cells can be measured using any suitable method known in the art,such as cytotoxicity assays described in, for example, Kochenderfer etal., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J.Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, thebiological activity of the cells also can be measured by assayingexpression and/or secretion of certain cytokines, such as CD 107a, IFNγ,IL-2, and TNF. In certain embodiments, the biological activity ismeasured by assessing clinical outcome, such as reduction in tumorburden or load. In certain embodiments, toxic outcomes, persistenceand/or expansion of the cells, and/or presence or absence of a hostimmune response, are assessed.

7.7. RADIATION THERAPY

Radiation therapies which are suitable for use in the combinationtreatments described herein, include, but not limited to, external beamradiation, a radiopharmaceutical agent which comprises aradiation-emitting radioisotope, internal radiation therapy,radionuclide therapy, and radiation surgery.

In certain embodiments, the presently disclosed combination methodsinclude use of external beam radiation therapy. External beam radiationtherapy uses a radiation (ionizing radiation) source that is external tothe subject (e.g., at the region of the subject that contains thelesion), typically either a radioisotope, such as ⁶⁰Co, ¹³⁷Cs, or a highenergy x-ray source, such as a linear accelerator. In certainembodiments, external beam radiation therapy comprises orthovoltage(i.e., superficial) beams of radiation to treat and/or disrupt a lesionpresent on the skin. In certain embodiments, external beam radiationtherapy comprises megavoltage, e.g., deep, beams of radiation are usedto treat internal lesions, e.g., lesions of the bladder, bowel,prostate, lung, or brain. In certain embodiments, external beamradiation therapy comprises delivering X rays, gamma rays, electronbeams, proton beams, or beams of ionized nuclei to the lesion. Incertain embodiments, the external beam radiation therapy is performedwith a linear accelerator, a collimator, a cobalt machine, a superficialradiation therapy (SRT) machine, Orthovoltage X ray machine.

The external source produces a collimated beam directed into the patientto the lesion (e.g., tumor) site. The adverse effect of irradiating ofhealthy tissue can be reduced, while maintaining a given dose ofradiation in the tumorous tissue, by projecting the external radiationbeam into the patient at a variety of “gantry” angles with the beamsconverging on the lesion (e.g., tumor) site. The particular volumeelements of healthy tissue, along the path of the radiation beam,change, reducing the total dose to each such element of healthy tissueduring the entire treatment. The irradiation of healthy tissue also canbe reduced by tightly collimating the radiation beam to the generalcross section of the tumor taken perpendicular to the axis of theradiation beam. Numerous systems exist for producing such acircumferential collimation, some of which use multiple sliding shutterswhich, piecewise, can generate a radio-opaque mask of arbitrary outline.

In certain embodiments, the presently disclosed combination methodsinclude administering to the subject a radiopharmaceutical agent. A“radiopharmaceutical agent”, as defined herein, refers to apharmaceutical agent which includes at least one radiation-emittingradioisotope. Radiopharmaceutical agents are routinely used in nuclearmedicine for the diagnosis and/or therapy of various diseases. Theradiolabelled pharmaceutical agent, for example, a radiolabelledantibody, includes a radioisotope (RI) that serves as the radiationsource.

As used herein, the term “radioisotope” includes metallic andnon-metallic radioisotopes. The radioisotope is chosen based on themedical application of the radiolabeled pharmaceutical agents. When theradioisotope is a metallic radioisotope, a chelator is typicallyemployed to bind the metallic radioisotope to the rest of the molecule.When the radioisotope is a non-metallic radioisotope, the non-metallicradioisotope is typically linked directly, or via a linker, to the restof the molecule.

As used herein, a “metallic radioisotope” is any suitable metallicradioisotope useful in a therapeutic or diagnostic procedure in vivo orin vitro. Suitable metallic radioisotopes include, but are not limitedto: Actinium-225, Antimony-124, Antimony-125, Arsenic-74, Barium-103,Barium-140, Beryllium-7, Bismuth-206, Bismuth-207, Bismuth 212, Bismuth213, Cadmium-109, Cadmium-115m, Calcium-45, Cerium-139, Cerium-141,Cerium-144, Cesium-137, Chromium-51, Cobalt-55, Cobalt-56, Cobalt-57,Cobalt-58, Cobalt-60, Cobalt-64, Copper-60, Copper-62, Copper-64,Copper-67, Erbium-169, Europium-152, Gallium-64, Gallium-67, Gallium-68,Gadolinium 153, Gadolinium-157 Gold-195, Gold-199, Hafnium-175,Hafnium-175-181, Holmium-166, Indium-110, Indium-111, Iridium-192, Iron55, Iron-59, Krypton 85, Lead-203, Lead-210, Lutetium-177, Manganese-54,Mercury-197, Mercury 203, Molybdenum-99, Neodymium-147, Neptunium-237,Nickel-63, Niobium 95, Osmium-185+191, Palladium-103, Palladium-109,Platinum-195m, Praseodymium-143, Promethium-147, Promethium-149,Protactinium-233, Radium-226, Rhenium-186, Rhenium-188, Rubidium-86,Ruthenium-97, Ruthenium-103, Ruthenium-105, Ruthenium-106, Samarium-153,Scandium-44, Scandium-46, Scandium-47, Selenium-75, Silver-110m,Silver-111, Sodium-22, Strontium-85, Strontium-89, Strontium-90,Sulfur-35, Tantalum-182, Technetium-99m, Tellurium-125, Tellurium-132,Thallium-204, Thorium-228, Thorium-232, Thallium-170, Tin-113, Tin-114,Tin-117m, Titanium-44, Tungsten-185, Vanadium-48, Vanadium-49,Ytterbium-169, Yttrium-86, Yttrium-88, Yttrium-90, Yttrium-91, Zinc-65,Zirconium-89, Zirconium-95, and Dysprosium-165.

As used herein, a “non-metallic radioisotope” is any suitablenonmetallic radioisotope (non-metallic radioisotope) useful in atherapeutic or diagnostic procedure in vivo or in vitro. Suitablenon-metallic radioisotopes include, but are not limited to: Iodine-131,Iodine-125, Iodine-123, Phosphorus-32, Astatine-211, Fluorine-18,Carbon-11, Oxygen-15, Bromine-76, and Nitrogen-13.

One of ordinary skill in the art may select a specific biomolecule foruse in targeting a particular neoplasm for radionuclide therapy basedupon the cell-surface molecules present on that neoplasm.

Identifying the most appropriate isotope for radiotherapy requiresweighing a variety of factors. These include tumor uptake and retention,blood clearance, rate of radiation delivery, half-life and specificactivity of the radioisotope, and the feasibility of large-scaleproduction of the radioisotope in an economical fashion. The key pointfor a therapeutic radiopharmaceutical is to deliver the requisite amountof radiation dose to the tumor cells and to achieve a cytotoxic ortumoricidal effect while not causing unmanageable side-effects.

In certain embodiments, the physical half-life of the therapeuticradioisotope is similar to the biological half-life of theradiopharmaceutical at the lesion (e.g., tumor) site. For example, ifthe half-life of the radioisotope is too short, much of the decay mayhave occurred before the radiopharmaceutical has reached maximumtarget/background ratio. On the other hand, too long a half-life maycause unnecessary radiation dose to normal tissues. Ideally, theradioisotope should have a long enough half-life to attain a minimumdose rate and to irradiate all the cells during the most radiationsensitive phases of the cell cycle. In addition, the half-life of aradioisotope has to be long enough to allow adequate time formanufacturing, release, and transportation.

Other practical considerations in selecting a radioisotope for a givenapplication in tumor therapy are availability and quality. The purityhas to be sufficient and reproducible, as trace amounts of impuritiescan affect the radiolabeling and radiochemical purity of theradiopharmaceutical.

The target receptor sites in tumors are typically limited in number. Assuch, in certain embodiments, the radioisotope have high specificactivity. The specific activity depends primarily on the productionmethod. Trace metal contaminants must be minimized as they often competewith the radioisotope for the chelator and their metal complexes competefor receptor binding with the radiolabeled chelated agent.

In certain embodiments, the radiation treatment used in the combinationmethods of the present disclosure is a combination of external beamradiation and a radioisotope, such as a radiopharmaceutical agent.

In certain embodiments, the presently disclosed combination methodsinclude use of internal radiation therapy, e.g., brachytherapy. Incertain embodiments, the brachytherapy comprises applying sources ofradiation at or near the area of the lesion. In certain embodiments, thebrachytherapy comprises interstitial radiation wherein the radiationsource is contained in small pellets, seeds, wires, tubes, and/orcontainers and is placed directly into or next to the lesion. In certainembodiments, the brachytherapy comprises intracavitary radiation,wherein a container of radioactive material is placed in a cavity of thebody, e.g., chest cavity or large intestine. In certain embodiments,ultrasounds, X-rays, and/or CT scans are used to assist with theplacement of the radioactive source.

In certain embodiments, the presently disclosed combination methodsinclude use of permanent brachytherapy, which comprises placing smallcontainers, e.g., containers approximately the size of a grain of rice,into a lesion. In certain embodiments, containers give off radiation fora time period of several weeks or months, and are left in place afterthe radiation is used up.

In certain embodiments, the presently disclosed combination methodsinclude use of temporary brachytherapy which comprises placingcylinders, hollow needles, tubes (catheters), and/or fluid-filledballoons into the area to be treated that are then removed aftertreatment. In certain embodiments, radioactive materials are placed inthese containers for a short time and then removed. In certainembodiments, the temporary brachytherapy can be high-dose rate (HDR)brachytherapy, wherein the radiation source is put into place for a fewminutes at a time at or near the lesion, and then is removed. Thisprocess may be repeated twice a day for up to a week, or once a week fora few weeks. In certain embodiments, the temporary brachytherapy is lowdose rate (LDR) brachytherapy, wherein the radiation source stays inplace for up to 7 days before it is removed.

The type of radiation that is suitable for use in the combinationmethods disclosed herein can vary. For example, radiation can beelectromagnetic or particulate in nature. Electromagnetic radiationuseful in the practice of the presently disclosed combination methodsincludes, but is not limited to, x-rays and gamma rays. Particulateradiation useful in the practice of the presently disclosed combinationmethods includes, but is not limited to, electron beams (betaparticles), protons beams, neutron beams, alpha particles, and negativepi mesons.

The radiation can be delivered using conventional radiological treatmentapparatus and methods, and by intraoperative and stereotactic methods.Additional discussion regarding radiation treatments suitable for use inthe practice of the presently disclosed combination methods can be foundthroughout Steven A. Leibel et al., Textbook of Radiation Oncology(1998); (publ. W. B. Saunders Company), and particularly in Chapters 13and 14. Radiation can also be delivered by other methods such astargeted delivery, for example by radioactive “seeds,” or by systemicdelivery of targeted radioactive conjugates. J.; Padawer et al.,Combined Treatment with Radioestradiol lucanthone in Mouse C3HBA MammaryAdenocarcinoma and with Estradiol lucanthone in an Estrogen Bioassay,Int. J. Radiat. Oncol. Biol. Phys. 7: 347-357 (1981).

For tumor therapy, both α and β-particle emitters have beeninvestigated. Alpha particles are particularly good cytotoxic agentsbecause they dissipate a large amount of energy within one or two celldiameters. The (β-particle emitters have relatively long penetrationrange (2-12 mm in the tissue) depending on the energy level. Thelong-range penetration is particularly important for solid tumors thathave heterogeneous blood flow and/or receptor expression. The(β-particle emitters yield a more homogeneous dose distribution evenwhen they are heterogeneously distributed within the target tissue.

7.8. ADMINISTRATION OF RADIATION THERAPY

The combination methods of the present disclosure include administeringto the subject an effective dose of radiation. The radiation can beprovided by targeted delivery. The radiation can also be provided bysystemic delivery (e.g., systemic delivery of targeted radioactiveconjugates, for example a radiolabeled antibody).

7.8.1. ADMINISTRATION OF EXTERNAL BEAM RADIATION

For administration of external beam radiation, the amount can be atleast about 1 Gray (Gy) fractions at least once every other day to atreatment volume at a lesion site. In certain embodiments, the radiationis administered in at least about 2 Gray (Gy) fractions at least onceper day (daily) to a treatment volume at a lesion site. In certainembodiments, the radiation is administered in at least about 4 Gray (Gy)fractions at least once per day (daily) to a treatment volume at alesion site. In certain embodiments, the radiation is administered in atleast about 2 Gray (Gy) fractions at least once per day to a treatmentvolume for five consecutive days per week at a lesion site. In certainembodiments, the radiation is administered in at least about 4 Gray (Gy)fractions at least once per day to a treatment volume for five days perweek at a lesion site. In certain embodiments, the radiation isadministered in about 4 Gray (Gy) fractions daily for five days at alesion site. In certain embodiments, the radiation is administered in atleast about 4 Gray (Gy) fractions once per day for five consecutive daysat a lesion site. In certain embodiments, radiation is administered in10 Gy fractions every other day, three times per week to a treatmentvolume at a lesion site.

In certain embodiments, a total of at least between about 5 Gy and about40 Gy (e.g., between about 5 Gy and about 30 Gy, between about 10 Gy andabout 30 Gy, between about 5 Gy and about 20 Gy, between about 10 Gy andabout 20 Gy, or between about 10 Gy and about 30 Gy) is administered toa lesion site of a subject in need thereof. In certain embodiments, atotal of at least about 10 Gy is administered to a lesion site of asubject in need thereof. In certain embodiments, a total of at leastabout 20 Gy is administered to a lesion site of a subject in needthereof. In certain embodiments, at least about 30 Gy is administered toa lesion site of a subject in need thereof. In certain embodiments, atleast about 40 Gy is administered to a lesion site of a subject in needthereof. In certain embodiments, a total of about 20 Gy is administeredto a lesion site of a subject in need thereof.

Typically, the subject receives external beam therapy four or five timesa week. An entire course of treatment usually lasts from one to sevenweeks depending on the type of cancer and the goal of treatment. Forexample, a subject can receive a dose of 2 Gy/day over 30 days.

7.8.2. ADMINISTRATION OF RADIOPHARMACEUTICAL AGENT

There are a number of methods for administration of aradiopharmaceutical agent. For example, the radiopharmaceutical agentcan be administered by targeted delivery or by systemic delivery oftargeted radioactive conjugates, such as a radiolabeled antibody, aradiolabeled peptide and a liposome delivery system.

In certain embodiments of targeted delivery, the radiolabelledpharmaceutical agent can be a radiolabelled antibody. See, for example,Ballangrud A. M., et al. Cancer Res., 2001; 61: 2008-2014 and Goldenber,D. M. J. Nucl. Med., 2002; 43 (5): 693-713, the contents of which areincorporated by reference herein. In certain embodiments of targeteddelivery, the radiopharmaceutical agent can be administered in the formof liposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines. See, for example, Emfietzoglou D, Kostarelos K,Sgouros G. An analytical dosimetry study for the use ofradionuclide-liposome conjugates in internal radiotherapy. J Nucl Med2001; 42: 499-504, the contents of which are incorporated by referenceherein.

In certain embodiments of targeted delivery, the radiolablledpharmaceutical agent can be a radiolabeled peptide. See, for example,Weiner R E, Thakur M L. Radiolabeled peptides in the diagnosis andtherapy of oncological diseases. Appl Radiat Isot 2002 November; 57 (5):749-63, the contents of which are incorporated by reference herein.

In addition to targeted delivery, Bracytherapy can be used to deliverthe radiopharmaceutical agent to the target site. Brachytherapy is atechnique that puts the radiation sources as close as possible to thelesion (e.g., tumor) site. Often the source is inserted directly intothe lesion (e.g., tumor). The radioactive sources can be in the form ofwires, seeds or rods. Generally, cesium, iridium or iodine are used.

The amount of radiation necessary can be determined by one of skill inthe art based on known doses for a particular type of the disease ordisorder (e.g., cancer). In certain embodiments, the radiation can beadministered in amount effective to cause the arrest or regression ofthe cancer of, when the radiation is administered with the geneticallyengineered cells to provide the unexpected synergistic effect, e.g., asynergistic abscopal-like response. HDAC inhibitor.

7.9. REDUCTION IN DISEASE BURDEN, EFFICACY AND SURVIVAL

The administration in accord with the presently disclosed methodsgenerally reduces or prevents the expansion or burden of the disease orcondition in the subject. For example, where the disease or condition isa tumor, the methods generally reduce tumor size, bulk, metastasis,percentage of blasts in the bone marrow or molecularly detectable cancerand/or improve prognosis or survival or other symptom associated withtumor burden.

Disease burden can encompass a total number of cells of the disease inthe subject or in an organ, tissue, or bodily fluid of the subject, suchas the organ or tissue of the tumor or another location, e.g., whichwould indicate metastasis. For example, tumor cells may be detectedand/or quantified in the blood or bone marrow in the context of certainhematological malignancies. In certain embodiments, disease burdenincludes, the mass of a tumor, the number or extent of metastases and/orthe percentage of blast cells present in the bone marrow.

In certain embodiments, the subject suffers from blood cancer. Incertain embodiments, the subject suffers from multiple myeloma (“MM”).The extent of disease burden can be determined by assessment of residualMM in blood or bone marrow, e.g., based on the International MyelomaWorking Group (IMWG) criteria for diagnosis for MM. In certainembodiments, the subject exhibits morphologic disease if a monoclonalparaprotein (M-spike) is detected (e.g., by serum proteinelectrophoresis (SPEP)). In certain embodiments, the subject exhibitsmorphologic disease if bone marrow infiltration by monoclonal malignantplasma cells (PC) is about 5% or greater. In certain embodiments, thesubject exhibits morphologic disease if bone-based and/or extra-osseousMM lesions are detected. In certain embodiments, the subject exhibitscomplete or clinical remission if M-spike is undetectable, bone marrowinfiltration by monoclonal malignant plasma cells (PC) is less thanabout 5%, and/or absence of bone-based and/or extra-osseous MM lesions.

In certain embodiments, the disease or condition persists followingadministration of the first dose and/or administration of the first doseis not sufficient to eradicate the disease or condition in the subject.

In certain embodiments, administration of the consecutive dose reducesdisease burden as compared to disease burden at a time immediately priorto the first dose, or at a time immediately prior to the consecutivedose. In certain embodiments, for example in the context of relapse,administration of the consecutive dose effects a reduction in diseaseburden as compared to the peak level of disease burden followingadministration of the first dose.

In certain embodiments, the method reduces the burden of the disease orcondition, e.g., number of tumor cells, size of tumor, duration ofpatient survival or event-free survival, to a greater degree and/or fora greater period of time as compared to the reduction that would beobserved with a comparable method using an alternative dosing regimen,such as one including administrating genetically engineered cellswithout radiation therapy.

In certain embodiments, the burden of a disease or condition in thesubject is detected, assessed, or measured. Disease burden may bedetected in certain embodiments by detecting the total number of diseaseor disease-associated cells, e.g., tumor cells, in the subject, or in anorgan, tissue, or bodily fluid of the subject, such as blood or serum.In certain embodiments, disease burden, e.g. tumor burden, is assessedby measuring the mass of a solid tumor and/or the number or extent ofmetastases. In certain embodiments, survival of the subject, survivalwithin a certain time period, extent of survival, presence or durationof event-free or symptom-free survival, or relapse-free survival, isassessed. In certain embodiments, any symptom of the disease orcondition is assessed. In certain embodiments, the measure of disease orcondition burden is specified.

In certain embodiments, the event-free survival rate or overall survivalrate of the subject is improved by the methods, as compared with othermethods. For example, in certain embodiments, event-free survival rateor probability for subjects treated by the methods at about five weeks,about six weeks, about seven weeks, about eight weeks, about threemonths, about four months, about five months, about six months followingthe first dose is greater than about 40%, greater than about 50%,greater than about 60%, greater than about 70%, greater than about 80%,greater than about 90%, or greater than about 95%. In certainembodiments, overall survival rate is greater than about 40%, greaterthan about 50%, greater than about 60%, greater than about 70%, greaterthan about 80%, greater than about 90%, or greater than about 95%. Incertain embodiments, the subject treated with the methods exhibitsevent-free survival, relapse-free survival, or survival to at leastabout five weeks, about six weeks, about seven weeks, about eight weeks,about three months, about four months, about five months, about sixmonths, at least about 1 year, about 2 years, about 3 years, about 4years, about 5 years, about 6 years, about 7 years, about 8 years, about9 years, or about 10 years. In certain embodiments, the time toprogression is improved, such as a time to progression of greater thanabout 6 months, at least about 1 year, about 2 years, about 3 years,about 4 years, about 5 years, about 6 years, about 7 years, about 8years, about 9 years, or about 10 years following the first dose.

In certain embodiments, following treatment by the method, theprobability of relapse is reduced as compared to other methods. Forexample, in certain embodiments, the probability of relapse at aboutfive weeks, about six weeks, about seven weeks, about eight weeks, aboutthree months, about four months, about five months, about six monthsfollowing the first dose is less than about 80%, less than about 70%,less than about 60%, less than about 50%, less than about 40%, less thanabout 30%, less than about 20%, or less than about 10% as compared toother methods, e.g., one including administrating genetically engineeredcells without radiation therapy.

7.10. KITS

Also provided are articles of manufacture, such as kits, which containone or more cell doses according to any of the provided embodiments,such as in any of the formulations or compositions described (which maybe present in one or more containers, such as bags or vials, with cellsin sufficient numbers or concentration for administration of anindividual dose or a portion thereof), and instructions and/or packagingmaterials or literature with instructions for the administration inaccordance with a method as provided herein. In certain embodiments, theinstructions indicate the caution or contraindication of any of theembodiments described.

8. EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the presently disclosed subjectmatter.

The present disclosure is not intended to be limited in scope to theparticular disclosed embodiments, which are provided, for example, toillustrate various aspects of the present disclosure. Variousmodifications to the compositions and methods described will becomeapparent from the description and teachings herein. Such variations maybe practiced without departing from the true scope and spirit of thedisclosure and are intended to fall within the scope of the presentdisclosure.

Example 1—BCMA-Targeted CAR T-Cell Therapy Plus Radiation Therapy forthe Treatment of Refractory Myeloma Reveals Potential Synergy Summary:

This Example presents a case of a patient with multiply-relapsed,refractory myeloma whose clinical course showed evidence of asynergistic abscopal-like response to CAR T-cell therapy and localizedradiation therapy (XRT). Shortly after receiving B-cell maturationantigen (BCMA)—targeted CAR T-cell therapy, the patient required urgenthigh-dose steroids and XRT for spinal cord compression. Despite thesteroids, the patient had a durable systemic response that could not beattributed to XRT alone. Post-XRT findings included a second wave offever and increased CRP and IL6, beginning 21 days post-CAR T cells,which is late for cytokine release syndrome (CRS) from CAR T-celltherapy alone on this trial. Given this response, which resembled CRS,immediately following XRT, changes in the patient's T-cell receptor(TCR) repertoire were investigated over 10 serial time points. ComparingT-cell diversity via Morisita's Overlap Indices (C_(D)), it wasdiscovered that, although the diversity was initially stable after CART-cell therapy compared with baseline (C_(D)=0.89-0.97, baseline vs 4time points post-CAR T cells), T-cell diversity changed after theconclusion of XRT, with >30% newly expanded TCRs (C_(D)=0.56-0.69,baseline vs 4 time points after XRT). These findings suggest potentialsynergy between radiation and CAR T-cell therapies resulting in anabscopal-like response.

Materials and Methods

Clinical protocol: The patient was treated on a trial evaluatingautologous BCMA-targeted CAR T cells (Trial registration ID:NCT03070327; trial conducted in accordance with US Common Rule; informedwritten consent obtained from patients). Gene modification wasaccomplished via a retrovirus encoding a CAR that consists of ahuman-derived anti-BCMA scFv, a CD8 transmembrane domain, the signalingdomains of 4-1BB and CD3ζ fused to a “self-cleaving” P2A element, and aseparate gene encoding a surrogate transduction marker (Smith et al.,Mol Ther. (2018 Jun. 6); 26(6):1447-1456). Under the trial protocol,patients underwent eukopheresis, and peripheral blood mononuclear cells(PBMCs) were frozen. At the appropriate time, PBMCs were thawed,selected and activated with CD3/CD28 beads, and expanded in the presenceof IL2. The patient was admitted for cyclophosphamide and fludarabine(300 mg/m2 and 30 mg/m2, respectively, ×3 consecutive days) forlymphodepletion prior to the administration of CAR T cells. This was a3×3 dose escalation study, and the patient presented was treated on doselevel 3, at a planned 450×10⁶ dose of CAR⁺ viable T cells.

Radiation therapy: External beam radiation therapy using 6MV photons wasdelivered using a linear accelerator (Varian Medical Systems, Palo Alto,Calif.). The target areas included the T1 to T8 vertebral bodies with aright-sided paraspinal tumor mass and the whole brain to the C2vertebral body). Conventional opposed fields were used (AP/PA for thethorax and opposed laterals for the whole brain fields). The total dosewas 2000 cGy in 5 daily fractions to each site.

Cytokine measurement: IL-1β, IL6, IL-10, and TNFα in peripheral serumwere detected by 4-plex microfluidic sandwich immunoassays performed onan Ella (Protein Simple; San Jose, Calif.). CRP was detected byautomated immunoturbidimetric assay performed on an Abbott ArchitectChemistry Analyzer (Abbott Laboratories; Chicago, Ill.). Ferritin wasdetected by automated two-site immunoenzymatic assay on a Tosoh AIA 2000(Tosoh Bioscience; Japan). D-dimer was detected by automatedimmunoturbidimetric assay on the Stago STA-R Max analyzer (Stago;France).

Flow cytometry: To stain for CAR T cells, anti-CD3 clone 7D-6(Thermo-Fisher; Waltham, Mass.), anti-CD8 clone 3B5 (Thermo-Fisher;Waltham, Mass.), and cetuximab (Eli Lilly, Indianapolis, Ind.)conjugated with a lightning link labeling kit (Innova Biosciences,Cambridge, UK) were used. Viability was ascertained with 7AAD exclusion(Thermo-Fisher; Waltham, Mass.). Flow cytometry was performed on a BDLSR II (BD biosciences; San Jose, Calif.), and analyzed with FlowJo(FlowJo LLC; Ashland, Oreg.).

TCR clonotype tracking: DNA was extracted via QIAmp Blood DNA mini kit(Qiagen; Hilden, G R) from PBMCs or bone marrow mononuclear cells.Clonotypes were monitored by TCR Vβ CDR3 sequencing via immunoSEQ assay(Adaptive Biotechnologies; Seattle, Wash.).

Results Clinical Evaluation

A 63-year-old African American woman was diagnosed with IgAλ multiplemyeloma (MM) with high-risk cytogenetics (amplification 1q21) in 2010.She received 8 lines of therapy including combinations withlenalidomide, bortezomib, pomalidomide, carfilzomib, daratumumab, andautologous stem cell transplant, each time with progression. Mostrecently her disease was refractory to therapy with dexamethasone,cyclophosphamide, etoposide, and cisplatin (DCEP). She enrolled on aclinical trial of BCMA-targeted CAR T-cell therapy including a 4-1BBco-stimulatory domain (Smith et al., Mol Ther. (2018 Jun. 6);26(6):1447-1456) (NCT03070327). Her clinical course is presented inFIGS. 1A-1F. Baseline findings indicated extensive disease, includingmonoclonal paraprotein (M-spike) of 2.26 g/dL, a baseline bone marrowbiopsy revealing 95% plasma cell infiltration, and a PET/CT scandemonstrating widespread bone-based and extra-osseous MM lesions,including extensive soft tissue and pleural-based masses (see FIG. 1A).

The patient received cyclophosphamide/fludarabine lymphodepletingchemotherapy followed by a single infusion of CAR T cells. Shortlythereafter, she exhibited signs of lower extremity weakness andconfusion. The patient remained awake and alert, however, mental statuschanges in addition to confusion manifested as intermittent aphasia,decreased short-term recall (could recall 0/3 objects), andattention/calculation (could not spell WORLD backwards). To evaluatethese neurologic findings an MM spine and brain were obtained whichrevealed spinal cord compression and multiple epidural masses causing acerebral mid-line shift (FIG. 2A). On day 5 post-CAR T cells, M-spikehad increased from baseline to 2.83 g/dL. Although her neurologicsymptoms resolved by this point, given the lesion compressing herthoracic spinal cord on imaging and increasing MM serologic markers,there was concern for progression of her cord compression. The patienttherefore received high-dose steroids (dexamethasone taper starting at10 mg every 12 hours for a total of 13 days) and palliative XRT to thethoracic spine (T1 to T8) followed by the whole brain to C2; deliveredbetween day 6-20 post-CAR T cells (2000 cGy in 5 fractions to each site,in series) (see FIGS. 1A-1F and 2B). Her Mill at 4 weeks showednear-resolution of disease at the sites of radiation therapy (see FIG.2A).

In addition to this response at the site of radiation therapy, afavorable systemic response was subsequently observed. Seven weekspost-CAR T cells, her M-spike was undetectable (FIG. 1B). PET/CT at 8weeks post-CAR T cells showed complete radiographic resolution ofdisease including innumerable sites outside the radiation field (FIG.1C). The patient's clinical response persisted through 9 monthspost-therapy.

Inflammatory Response

Changes in serum inflammatory markers CRP, Ferritin, and D-dimer, aswell as cytokines IL-6, IL-10, TNFα, and IL-10, were prospectivelymonitored. Inflammatory markers associated with CAR T-cell activity(Davila et al., Sci Transl Med (2014); 6:224ra25) increased shortlyafter the conclusion of radiation therapy. These included markerscharacteristic of CAR T cell mediated cytokine release syndrome (CRS)(17): IL-6 (to 333 pg/mL from 32 pg/mL) (see FIG. 1D) and CRP (to 22.11mg/dL from 6.43 mg/dL) (see FIG. 1D), as well as more general markers ofinflammation that are often seen elevated in CRS: Ferritin (to 13,197ng/mL from 2,921 ng/mL (see FIG. 3A) and D-Dimer (to 915 ng/mL from <150ng/mL) (see FIG. 3B). Serum IL-10 had a bi-modal peak. The first peakoccurred early after the administration of CAR T cells, corresponding totiming of peak serum IL-10 concentrations after CAR T cells reported byothers (Kochenderfer et al., J Clin Oncol (2017); 35:1803-13) and thetiming of CRS seen in other patients on this study (Mailankody et al.,American Society of Hematology (2018); 132:959-959). A later second peakoccurred during radiation therapy (see FIG. 3C). TNFα and IL-10 remainedstable and within normal limits throughout the clinical course (see FIG.3C). Around the conclusion of radiation therapy, the patient developed afever to >39° C. with no clinical or laboratory evidence of infection(see FIG. 1E). At this time, she became tachycardic to 110 s beats/min(from recent baseline low 60 s beats/min) and SBP decreased to 90 s mmHg(from recent baseline 110-130 s mmHg); HR and BP returned to baselineafter 3 days of tachycardia and relative hypotension. She did notrequire vasopressors or supplemental oxygen, therefore this would beconsidered grade 1 CRS. A delayed CRS-like response was only seen inthis patient and not in other patients on the trial (n=13). Despite 13days of systemic steroids, CART cells expanded and persisted (see FIG.4), representing 9.3% of CD3⁺ cells in the blood by flow cytometryaround the time of inflammatory response (see FIG. 5). Given thetemporal relationship of these CRS-like findings immediately afterradiation therapy, the timing of changes in her T-cell receptor (TCR)repertoire was investigated using stored samples.

TCR Clonotype Serial Analysis

Because the post-radiation findings were suggestive of clinical andlaboratory findings often correlating with cytokine release syndrome(CRS), the timing of changes in the patient's T-cell receptor (TCR)repertoire was investigated using stored samples. It was found that thepatient's baseline peripheral blood TCR diversity mirrored that in herbaseline bone marrow (see FIG. 6; TCR diversity compared by Morisita'sOverlap Index; CD=0.97). Given this high degree of overlap betweenperipheral blood and bone marrow, it was concluded that the peripheralblood may be a suitable surrogate to investigate changes in TCRdiversity over time in this patient with intra- and extra-osseous MM.Serial analysis of the patient's peripheral blood TCR repertoiredemonstrated TCR diversity was stable at four time points across thefirst week after CAR T-cell infusion. Values for CD compared to baselinewere 0.89-0.97 throughout this time (see FIG. 7). A spike in new TCRclone expansion was noted upon initial evaluation, day eight after theconclusion of radiation therapy (C_(D)=0.56-0.69, 4-19 weeks post-CART-cell therapy vs baseline). These newly expanded clones made up >30% ofthe T-cell repertoire at 4 weeks. Many persisted through at least 19weeks post CAR T-cell infusion, the most recent time point assessed (seeFIG. 1F). No TCR clone expansion was seen in any clones highly expressedat baseline (see FIG. 8).

DISCUSSION

This Example reports a patient receiving CAR T-cell therapy forrefractory MM who received urgent high-dose steroids and palliative XRTto the whole brain and thoracic spine days later. The results illustratehow BCMA-targeted CAR T-cell therapy can facilitate elimination of alarge MM burden (including substantial extra-osseous disease) despitethe early and continued administration of high-dose steroids.Furthermore, the timing of CRS-like clinical signs and inflammatorymarkers coincided with the expansion of new TCR clones after radiationtherapy, supporting a synergistic effect between XRT and CAR T-celltherapy. The patient's clinical course suggests that local XRT incombination with CAR T-cell therapy may enhance a systemic anti-tumoreffect.

For frequent, serial assessment of TCR clonality, peripheral bloodmononuclear cells were relied. Despite the fact that MM is a bone marrowbased malignancy, and although marrow is accessible, bone marrow cannotbe sampled as frequently as can peripheral blood. It was found that,when assessed at the same time point, the patient's bone marrowmononuclear cells and PBMCs had a high degree of overlap in TCRrepertoires (C_(D)=0.97). Therefore, it was concluded that TCR clonalityin the peripheral blood is relevant in this patient with widespread MM.

Serial analysis of TCR repertoires revealed expansion of new TCR clonescoinciding with clinical and laboratory signs of CRS. The confluence ofthese events shortly after radiation therapy in this previously CART-cell treated patient supports the likelihood of synergy betweenradiation and CAR T-cell therapy.

One limitation of this study is that the stored material was not ofsufficient quantity to determine the antigen targets driving thespecific TCR clone expansion. However, newly expanded TCR clonescomprised >30% of the T-cell population after XRT at a time when CAR′ Tcells constituted ˜10% of CD3 cells, indicating that at least someT-cell expansion was driven by non-CAR modified T cells.

Radiation therapy, alone or in combination with checkpoint blockade, isknown to “shape” the TCR repertoire of expanded clones (Sridharan Br JCancer (2016); 115:252-60; Twyman-Saint et al., Nature (2015);520:373-7). However, given that CARs result in HLA-independent T-cellactivation, it is not necessarily intuitive that local radiation therapywould buoy systemic responses by CAR T cells in the way that it can buoycheckpoint blockade, i.e. through increased neo-antigen exposure throughan abscopal effect (Postow et al., N Engl J Med. (2012); 366:925-31).

Synergy of CAR T-cell and radiation therapy may occur through severalpossible mechanisms: (1) Synergy could be explained by the cytokinessecreted by CAR T-cells, increasing the likelihood of endogenous T-cellsmounting an abscopal-like response. (2) As has been shownpre-clinically, radiation therapy may enhance effector functions andmigration of CAR T-cells (Weiss et al., Clin Cancer Res (2018);24:882-95; DeSelm et al., Mol Ther (2018); 26:2542-52). (3) Increasedsignaling through the TCR of CAR T-cells may enhance clonal CAR T-cellexpansion. Combinations of these mechanisms or unknown mechanisms arealso possible explanations.

A similar phenomenon may have been at work in a CAR T cell-treatedpatient with CNS lymphoma for whom, after relapse, biopsy of a massprecipitated a clinical remission (Abramson et al., N Engl J Med (2017);377:783-4). These observations support further investigation of CART-cell therapy combined with radiation therapy.

Embodiments of the Presently Disclosed Subject Matter

From the foregoing description, it will be apparent that variations andmodifications may be made to the presently disclosed subject matter toadopt it to various usages and conditions. Such embodiments are alsowithin the scope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or sub-combination) of listed elements. The recitation ofan embodiment herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A method of treatment, comprising: (a)administering to a subject having a disease or condition a dose of cellsexpressing a recombinant receptor that binds to an antigen; and (b)administering to said subject radiation, wherein initiation ofadministration of the radiation is no later than about two weeks afteradministration of the recombinant receptor-expressing cells.
 2. Themethod of claim 1, wherein initiation of administration of the radiationis no later than about one week after administration of the recombinantreceptor-expressing cells.
 3. The method of claim 1, wherein initiationof administration of the radiation is between about 5 days and about 10days after administration of the recombinant receptor-expressing cells.4. The method of claim 1, wherein the subject has not relapsed at thetime of or immediately prior to initiation of administration of theradiation.
 5. The method of claim 1, wherein the disease or condition isa tumor or a cancer.
 6. The method of claim 1, wherein the disease orcondition is selected from the group consisting of blood cancers, B cellmalignancies, colon cancer, lung cancer, liver cancer, breast cancer,prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer,brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma,pancreatic adenocarcinoma, cervical carcinoma, colorectal cancer,glioblastoma, neuroblastoma, Ewing sarcoma, medulloblastoma,osteosarcoma, synovial sarcoma, mesothelioma, and combinations thereof.7. The method of claim 6, wherein the blood cancer is selected from thegroup consisting of leukemia, lymphoma, chronic lymphocytic leukemia(CLL), acute-lymphoblastic leukemia (ALL), Hodgkin Lymphoma,non-Hodgkin's lymphoma, Waldenstrom's Macroglobulinemia, acute myeloidleukemia, multiple myeloma, mantle cell lymphoma, and indolent B celllymphoma.
 8. The method of claim 6, wherein the disease or condition ismultiple myeloma.
 9. The method of claim 1, wherein the antigen is atumor antigen.
 10. The method of claim 9, wherein the tumor antigen isselected from the group consisting of BCMA, GPRC5D, FcRL5, orphantyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22,mesothelin, CEA, epatitis B surface antigen, anti-folate receptor, CD23,CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, OEPHa2, Erb-B2,Erb-B3, Erb-B4, FBP, fetal acetylcholine receptor (AChR), GD2, GD3,HMW-MAA, IL-22R-alpha, IL-13R-alpha2, KDR, kappa light chain, Lewis Y,L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2DLigands, NY-ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72,VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen,PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2,CD123, c-Met, GD-2, MAGE A3, CE7, Wilms Tumor 1 (WT-1), a cyclin, andbiotinylated molecules.
 11. The method of claim 10, wherein the tumorantigen is BCMA.
 12. The method of claim 1, wherein the recombinantreceptor is a T cell receptor (TCR) or a functional non-T cell receptor.13. The method of claim 12, wherein the recombinant receptor is achimeric antigen receptor (CAR).
 14. The method of claim 13, wherein theCAR comprises an extracellular antigen-binding domain that specificallybinds to the antigen and an intracellular signaling domain.
 15. Themethod of claim 14, wherein the intracellular signaling domain comprisesan intracellular domain of a CD3-zeta (CD3) chain.
 16. The method ofclaim 14, wherein the intracellular signaling domain further comprises acostimulatory signaling region.
 17. The method of claim 16, wherein thecostimulatory signaling region comprises a signaling domain of CD28 or aportion thereof, a signaling domain of 4-1BB or a portion thereof, asignaling domain of OX40 or a portion thereof, a signaling domain ofICOS or a portion thereof, a signaling domain of DAP-10 or a portionthereof, or a combination thereof
 18. The method of claim 17, whereinthe costimulatory signaling region comprises a signaling domain of 4-1BBor a portion thereof.
 19. The method of claim 13, wherein the CARfurther comprises a transmembrane domain.
 20. The method of claim 19,wherein the transmembrane domain comprises a transmembrane domain of CD8or a portion thereof, or a transmembrane domain of CD28 or a portionthereof.
 21. The method of claim 20, wherein the transmembrane domaincomprises a transmembrane domain of CD8 or a portion thereof.
 22. Themethod of claim 14, wherein the extracellular antigen-binding domaincomprises a scFv.
 23. The method of claim 14, wherein the extracellularantigen-binding domain comprises: a heavy chain variable region(“V_(H)”) CDR1 comprising the amino acid sequence set forth in SEQ IDNO: 1; a V_(H) CDR2 comprising the amino acid sequence set forth in SEQID NO: 2; a V_(H) CDR3 comprising the amino acid sequence set forth inSEQ ID NO: 3; a light chain variable region (“V_(L)”) CDR1 comprisingthe amino acid sequence set forth in SEQ ID NO: 4; a V_(L) CDR2comprising the amino acid sequence set forth in SEQ ID NO: 5; and aV_(L) CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.24. The method of claim 1, wherein the cell is a T cell.
 25. The methodof claim 24, wherein the T cell is selected from the group consisting ofa cytotoxic T lymphocyte (CTL), a regulatory T cell, atumor-infiltrating lymphocyte (TIL), and a Natural Killer T (NKT) cell.26. The method of claim 1, wherein the cell is autologous or allogenicto the subject.
 27. The method of claim 1, wherein the dose of cellscomprises cells in an amount sufficient for reduction in burden of adisease or condition in the subject.
 28. The method of claim 1,comprising administering to the subject a consecutive dose of therecombinant receptor-expressing cells after administration of a firstdose of the recombinant receptor-expressing cells.
 29. The method ofclaim 1, wherein the radiation is selected from the group consisting ofexternal beam radiation, a radiopharmaceutical agent, and a combinationthereof.
 30. The method of claim 1, wherein the radiation is externalbeam radiation.
 31. The method of claim 1, wherein a total of at leastabout 10 Gy of radiation is administered to a lesion site of thesubject.
 32. The method of claim 31, wherein a total of between about 10Gy and about 30 Gy of radiation is administered to a lesion site of thesubject.
 33. The method of claim 32, wherein a total of about 20 Gy ofradiation is administered to a lesion site of the subject.
 34. Themethod of claim 1, wherein administration of the radiation andadministration of the recombinant receptor-expressing cells provide asynergistic abscopal effect, delayed or reduced CRS-like response,and/or systemic expansion of new T-cell receptor (TCR) clone.